Microbial-based process for high-quality protein concentrate

ABSTRACT

The present invention describes a bio-based process to produce high quality protein concentrate (HQPC) by converting plant derived celluloses into bioavailable protein via aerobic incubation, including the use of such HQPC so produced as a nutrient, including use as a fish meal replacement in aquaculture diets.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 16/918,831, filedJul. 1, 2020 under 35 U.S.C. § 120, which is a continuation of U.S. Ser.No. 15/151,599, filed May 11, 2016 under 35 U.S.C. § 120, which is acontinuation of U.S. Ser. No. 13/691,843, filed Dec. 2, 2012 under 35U.S.C. § 120, now U.S. Pat. No. 9,370,200, which claims benefit under 35U.S.C. § 119(e) to U.S. Provisional Application No. 61/556,487, filed onDec. 2, 2011, and U.S. Provisional Application No. 61/566,557, filed onDec. 2, 2011, each of which is incorporated by reference herein in itsentirety.

BACKGROUND OF THE INVENTION

This work was made with Governmental support from the National ScienceFoundation under contract DBI-1005068. The Government has certain rightsin this invention.

FIELD OF THE INVENTION

The invention generally relates to incubation processes, andspecifically microbial-based aerobic incubation processes to producehigh quality protein concentrates, including products made therefrom anduse of such products in the formulation of nutrient feeds.

BACKGROUND INFORMATION

In 2008, approximately 28% of the world's wild, marine fish stocks wereoverexploited and 52% were fully exploited, even as the demand for percapita consumption of fish and shellfish products have increased withthe increasing human population. With dwindling wild fish stocks, in aneffort to meet this increased demand, commercial aquaculture productionhas increased dramatically. However, one of the primary constituents ofdietary formulations for aquaculture, fish meal protein, is also derivedfrom wild capture fisheries. It is estimated that at least 6.7 mmt offish meal will be required to support commercial aquaculture productionby 2012. This is clearly an unsustainable trend.

Lower cost, more sustainable plant-derived sources of protein have beenused to partially replace fish meal in aquaculture diets. Defattedsoybean meal (SBM, 42-48% protein) has commonly been used to replace upto 20% of total protein in grower diets for several species, while soyprotein concentrate (SPC, 65% protein) has been tested successfully athigher total protein replacement levels, largely governed by the trophicstatus of the species. These soybean products provide high protein andrelative good amino acid profiles, but are still deficient in somecritical amino acids (e.g., taurine) required by carnivorous marinefishes. SPC can be used at higher levels than soybean meal, primarilybecause the solvent extraction process used to produce SPC removesanti-nutritional factors (e.g., oligosaccharides) and thereby increasesprotein bioavailability. In addition, a thermal step has been used toinactivate heat-labile antigenic factors. The primary limitations of thecurrent solvent extraction process are its cost, the lack of use for theoligosaccharides removed in the process, and quality issues thatfrequently limit inclusion to 50% of total protein in the diet. Further,processing of soy material into soybean meal or soy protein concentratescan be environmentally problematic (e.g., problems with disposal ofchemical waste associated with hexane-extraction).

Corn co-products, including dried distiller's grains with solubles(DDGS), have also been evaluated in aquaculture diets at fish mealreplacement levels of up to 20%. DDGS has lower protein (28-32%) andmore fiber than soy products, but is typically priced at ˜50% of thevalue of defatted soybean meal. Some ethanol plants have incorporated adry fractionation process to remove part of the fiber and oil prior tothe conversion process, resulting in a dry-frac DDGS of up to 42%protein. While this product has been used to replace 20-40% of fish mealin aquaculture feeds, there remains the need for a higher protein, moredigestible DDGS aqua feed product. Such a product would be especiallyattractive if the protein component had higher levels of critical aminoacids such as taurine, lysine, methionine, and cysteine.

Therefore, a plant-derived protein source which is cost-effective and“green,” and that is of a high-enough quality to fully or substantiallyreplace more of the fish meal in an aquaculture diet is needed.

SUMMARY OF THE INVENTION

The present disclosure relates to an organic, microbially-based systemto convert plant material into a highly digestible, concentrated proteinsource that also contains a microbial gum (exopolysaccharide) binder,including such a concentrated source which is suitable for use as a feedfor animals used for human consumption.

In embodiments, a composition containing a non-animal based proteinconcentrate is disclosed, where the composition contains at least 55%protein content and no detectable stachyose on a dry matter basis. Inone aspect, the composition contains Aureobasidium pullulans depositedstrain NRRL No. 50792, NRRL No. 50793, NRRL No. 50794, NRRL No. 50795,or a combination thereof.

In one aspect, the non-animal based protein concentrate is isolated fromcereal grain and oilseed plant material including, but not limited to,soybeans, peanuts, Rapeseeds, canola, sesame seeds, barley, cottonseeds,palm kernels, grape seeds, olives, safflowers, sunflowers, copra, corn,coconuts, linseed, hazelnuts, wheat, rice, potatoes, cassavas, legumes,camelina seeds, mustard seeds, germ meal, corn gluten meal,distillery/brewery by-products, portions and combinations thereof.

In another aspect, the protein content of the composition is in therange of from about 56% to about 90% on a dry matter basis produced by aprocess including extruding plant material at above room temperature toform a mash; adding one or more cellulose-deconstructing enzymes torelease sugars into the mash; inoculating the enzyme-treated mash withat least one microbe, which microbe converts released sugars intoproteins and exopolysaccharides; precipitating the resulting proteins,microbes, and exopolysaccharides with ethanol or a flocculent;recovering the precipitated material via hydrodynamic force; and dryingsaid precipitated material.

In a related aspect, the at least one microbe includes, but is notlimited to, Aureobasidium pullulans, Sclerotium glucanicum, Sphingomonaspaucimobilis, Ralstonia eutropha, Rhodospirillum rubrum, Kluyveromycesspp, Pichia spp, Trichoderma reesei, Pleurotus ostreatus, Rhizopus spp,and combinations thereof.

In one aspect, the plant material is from soybeans in the form of soyflakes or soy meal. In another aspect, the plant material is from oilseeds or their de-oiled meals. In another aspect, the plant material isfrom distiller's dried grain with solubles (DDGS).

In one aspect, the protein concentrate includes at least 0.1 ghydroxylysine/100 g of concentrate.

In one embodiment, an animal feed comprising a non-animal based proteinconcentrate is disclosed, where the composition includes at least about1.25 g of lipid/100 g composition, where the composition contains nodetectable stachyose or raffinose and at least 55% protein content on adry matter basis, and where the composition includes at least 35% ofsaid animal feed by weight.

In a related aspect, the composition is a complete replacement foranimal-based fishmeal in a fish feed. In a further related aspect, fishfeed is formulated for fish including, but not limited to, Siberiansturgeon, Sterlet sturgeon, Starry sturgeon, White sturgeon, Arapaima,Japanese eel, American eel, Short-finned eel, Long-finned eel, Europeaneel, Chanos chanos, Milkfish, Bluegill sunfish, Green sunfish, Whitecrappie, Black crappie, Asp, Catla, Goldfish, Crucian carp, Mud carp,Mrigal carp, Grass carp, Common carp, Silver carp, Bighead carp,Orangefin labeo, Roho labeo, Hoven's carp, Wuchang bream, Black carp,Golden shiner, Nilem carp, White amur bream, Thai silver barb, Java,Roach, Tench, Pond loach, Bocachico, Dorada, Cachama, Cachama Blanca,Paco, Black bullhead, Channel catfish, Bagrid catfish, Blue catfish,Wels catfish, Pangasius (Swai, Tra, Basa) catfish, Striped catfish,Mudfish, Philippine catfish, Hong Kong catfish, North African catfish,Bighead catfish, Sampa, South American catfish, Atipa, Northern pike,Ayu sweetfish, Vendace, Whitefish, Pink salmon, Chum salmon, Cohosalmon, Masu salmon, Rainbow trout, Sockeye salmon, Chinook salmon,Atlantic salmon, Sea trout, Arctic char, Brook trout, Lake trout,Atlantic cod, Pejerrey, Lai, Common snook, Barramundi/Asian sea bass,Nile perch, Murray cod, Golden perch, Striped bass, White bass, Europeanseabass, Hong Kong grouper, Areolate grouper, Greasy grouper, Spottedcoralgrouper, Silver perch, White perch, Jade perch, Largemouth bass,Smallmouth bass, European perch, Zander (Pike-perch), Yellow Perch,Sauger, Walleye, Bluefish, Greater amberjack, Japanese amberjack,Snubnose pompano, Florida pompano, Palometa pompano, Japanese jackmackerel, Cobia, Mangrove red snapper, Yellowtail snapper, Darkseabream, White seabream, Crimson seabream, Red seabream, Red porgy,Goldlined seabream, Gilthead seabream, Red drum, Green terror, Blackbeltcichlid, Jaguar guapote, Mexican mojarra, Pearlspot, Three spottedtilapia, Blue tilapia, Longfin tilapia, Mozambique tilapia, Niletilapia, Tilapia, Wami tilapia, Blackchin tilapia, Redbreast tilapia,Redbelly tilapia, Golden grey mullet, Largescale mullet, Gold-spotmullet, Thinlip grey mullet, Leaping mullet, Tade mullet, Flathead greymullet, White mullet, Lebranche mullet, Pacific fat sleeper, Marblegoby, White-spotted spinefoot, Goldlined spinefoot, Marbled spinefoot,Southern bluefin tuna, Northern bluefin tuna, Climbing perch, Snakeskingourami, Kissing gourami, Giant gourami, Snakehead, Indonesiansnakehead, Spotted snakehead, Striped snakehead, Turbot, Bastard halibut(Japanese flounder), Summer Flounder, Southern flounder, Winterflounder, Atlantic Halibut, Greenback flounder, Common sole, andcombinations thereof.

In one aspect, the fish feed effects greater performance in one or moreperformance aspects including, but not limited to, growth, weight gain,protein efficiency ratio, feed conversion ratio, total consumption,survival, and Fulton's condition factor compared to equivalent fish feedcomprising animal-based fishmeal or soy protein concentrate.

In another aspect, the fish feed effects the performance aspects at acrude protein content that is less than or equal to the protein contentof equivalent fish feed comprising animal-based fishmeal or soy proteinconcentrate.

In one aspect, the animal feed is supplemented with lysine, methionine,lipids, biotin, choline, niacin, ascorbic acid, inositol, pantothenicacid, folic acid, pyridoxine, riboflavin, thiamin, vitamin A, vitaminB12, vitamin D, vitamin E, vitamin K, calcium, phosphorus, potassium,sodium, magnesium, manganese, aluminum, iodine, cobalt, zinc, iron,selenium or a combination thereof.

In another embodiment, a method of producing a non-animal based proteinconcentrate is disclosed including extruding plant material at aboveroom temperature to form a mash and transferring the mash to abiorector; adding one or more cellulose-deconstructing enzymes torelease sugars into the mash in the bioreactor; inoculating the enzymetreated mash with at least one microbe, which microbe converts releasedsugars into proteins and exopolysaccharides; precipitating the resultingproteins, microbes, and exopolysaccharides with ethanol, a flocculent ora combination thereof; recovering the precipitated material viahydrodynamic force; and drying the precipitated material.

In a related aspect, extrusion is carried out at between about 50° C. toabout 170° C., at a compression ratio of about 3:1, and at a screw speedsufficient to provide a shearing effect against ridged channels on bothsides of an extrusion barrel.

In another related aspect, the method includes mixing the extrudedmaterials with water to achieve a solid loading rate of at least 5% inthe bioreactor; and optionally, autoclaving and cooling the dilutedextruded materials, where the one or more cellulose-deconstructingenzymes are selected from the group consisting of endo-xylanase andbeta-xylosidase, glycoside hydrolase, ß-glucosidases, hemicellulaseactivities, and combinations thereof.

In one related aspect, the method includes reducing the temperature ofthe enzyme treated mash to between about 30° C. to about 37° C.;inoculating the cooled mash with 2% (v/v) of a 24 hour culture of the atleast one microbe, where the at least one microbe includes, but is notlimited to Aureobasidium pullulans, Sclerotium glucanicum, Sphingomonaspaucimobilis, Ralstonia eutropha, Rhodospirillum rubrum, Kluyveromycesspp, Pichia spp, Trichoderma reesei, Pleurotus ostreatus, Rhizopus spp,and combinations thereof; optionally aerating the inoculated mash atabout 0.05 L/L/min; and incubating until utilization of sugars ceases orafter about 96 to 120 hours incubation in the presence of the at leastone microbe.

In one aspect, the method includes adding about 0.6 L ethanol/L of mash;centrifuging the ethanol treated mash; recovering the ethanol;optionally recovering fine suspended particles, recovering centrifugedsolids; and drying the recovered centrifuge solids. In another aspect,the supernatant may be dried, dried solids recovered, and thereaftermixed with the centrifuge solids.

In one embodiment, a biologically pure culture of Aureobasidiumpullulans strain selected from the group consisting of NRRL No. 50792,NRRL No. 50793, NRRL No. 50794, and NRRL No. 50795 is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow chart for the HQSPC conversion process.

FIG. 2 shows a flow chart for the HQSPC conversion process for aquafeeds.

FIG. 3 shows bench scale, extended incubation trials to evaluate HQSPCcomposition and yield.

FIG. 4 shows a flow chart for the HP-DDGS conversion process for aquafeeds.

FIG. 5 shows the effect of moisture content and extrusion speed onglucose recovery following extrusion of HP-DDGS at 100° C.

DETAILED DESCRIPTION OF THE INVENTION

Before the present composition, methods, and methodologies aredescribed, it is to be understood that this invention is not limited toparticular compositions, methods, and experimental conditions described,as such compositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “a nucleicacid” includes one or more nucleic acids, and/or compositions of thetype described herein which will become apparent to those personsskilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, as it will be understood thatmodifications and variations are encompassed within the spirit and scopeof the instant disclosure.

As used herein, “about,” “approximately,” “substantially” and“significantly” will be understood by a person of ordinary skill in theart and will vary in some extent depending on the context in which theyare used. If there are uses of the term which are not clear to personsof ordinary skill in the art given the context in which it is used,“about” and “approximately” will mean plus or minus <10% of particularterm and “substantially” and “significantly” will mean plus orminus >10% of the particular term.

As used herein, the term “animal” means any organism belonging to thekingdom Animalia and includes, without limitation, humans, birds (e.g.poultry), mammals (e.g. cattle, swine, goal, sheep, cat, dog, mouse andhorse) as well as aquaculture organisms such as fish (e.g. trout,salmon, perch), mollusks (e.g. clams) and crustaceans (e.g. lobster andshrimp).

Use of the term “fish” includes all vertebrate fish, which may be bonyor cartilaginous fish.

As used herein “non-animal based protein” means that the proteinconcentrate comprises at least 0.81 g of crude fiber/100 g ofcomposition (dry matter basis), which crude fiber is chiefly celluloseand lignin material obtained as a residue in the chemical analysis ofvegetable substances.

As used herein, “incubation process” means the provision of properconditions for growth and development of bacteria or cells, where suchbacteria or cells use biosynthetic pathways to metabolize various feedstocks. In embodiments, the incubation process may be carried out, forexample, under aerobic conditions. In other embodiments, the incubationprocess may include fermentation.

As used herein, the term “incubation products” means any residualsubstances directly resulting from an incubation process/reaction. Insome instances, an incubation product contains microorganisms such thatit has a nutritional content enhanced as compared to an incubationproduct that is deficient in such microorganisms. The incubationproducts may contain suitable constituent(s) from an incubation broth.For example, the incubation products may include dissolved and/orsuspended constituents from an incubation broth. The suspendedconstituents may include undissolved soluble constituents (e.g., wherethe solution is supersaturated with one or more components) and/orinsoluble materials present in the incubation broth. The incubationproducts may include substantially all of the dry solids present at theend of an incubation (e.g., by spray drying an incubation broth and thebiomass produced by the incubation) or may include a portion thereof.The incubation products may include crude material from incubation wherea microorganism may be fractionated and/or partially purified toincrease the nutrient content of the material.

As used herein, a “conversion culture” means a culture of microorganismswhich are contained in a medium that comprises material sufficient forthe growth of the microorganisms, e.g., water and nutrients. The term“nutrient” means any substance with nutritional value. It can be part ofan animal feed or food supplement for an animal. Exemplary nutrientsinclude but are not limited to proteins, peptides, fats, fatty acids,lipids, water and fat soluble vitamins, essential amino acids,carbohydrates, sterols, enzymes and trace minerals, such as, phosphorus,iron, copper, zinc, manganese, magnesium, cobalt, iodine, selenium,molybdenum, nickel, fluorine, vanadium, tin, and silicon.

Conversion is the process of culturing microorganisms in a conversionculture under conditions suitable to convertprotein/carbohydrate/polysaccharide materials, for example, soybeanmaterial into a high-quality protein concentrate. Adequate conversionmeans utilization of 90% or more of specified carbohydrates to producemicrobial cell mass and/or exopolysaccharide. In embodiments, conversionmay be aerobic or anaerobic.

As used herein a “flocculent” or “clearing agent” is a chemical thatpromotes colloids to come out of suspension through aggregation, andincludes, but is not limited to, a multivalent ion and polymer. Inembodiments, such a flocculent/clearing agent may include bioflocculentssuch as exopolysaccharides.

A large number of plant protein sources may be used in connection withthe present disclosure as feed stocks for conversion. The main reasonfor using plant proteins in the feed industry is to replace moreexpensive protein sources, like animal protein sources. Anotherimportant factor is the danger of transmitting diseases through feedinganimal proteins to animals of the same or related species. Examples forplant protein sources include, but are not limited to, protein from theplant family Fabaceae as exemplified by soybean and peanut, from theplant family Brassiciaceae as exemplified by canola, cottonseed, theplant family Asteraceae including, but not limited to sunflower, and theplant family Arecaceae including copra. These protein sources, alsocommonly defined as oilseed proteins may be fed whole, but they are morecommonly fed as a by-product after oils have been removed. Other plantprotein sources include plant protein sources from the family Poaceae,also known as Gramineae, like cereals and grains especially corn, wheatand rice or other staple crops such as potato, cassava, and legumes(peas and beans), some milling by-products including germ meal or corngluten meal, or distillery/brewery by-products. In embodiments, feedstocks for proteins include, but are not limited to, plant materialsfrom soybeans, peanuts, Rapeseeds, barley, canola, sesame seeds,cottonseeds, palm kernels, grape seeds, olives, safflowers, sunflowers,copra, corn, coconuts, linseed, hazelnuts, wheat, rice, potatoes,cassavas, legumes, camelina seeds, mustard seeds, germ meal, corn glutenmeal, distillery/brewery by-products, and combinations thereof.

In the fish farming industry the major fishmeal replacers with plantorigin reportedly used, include, but are not limited to, soybean meal(SBM), maize gluten meal, Rapeseed/canola (Brassica sp.) meal, lupin(Lupinus sp. like the proteins in kernel meals of de-hulled white(Lupinus albus), sweet (L. angustifolius) and yellow (L. luteus) lupins,Sunflower (Helianthus annuus) seed meal, crystalline amino acids; aswell as pea meal (Pisum sativum), Cottonseed (Gossypium sp.) meal,Peanut (groundnut; Arachis hypogaea) meal and oilcake, soybean proteinconcentrate, corn (Zea mays) gluten meal and wheat (Triticum aestivum)gluten, Potato (Solanum tuberosum L.) protein concentrate as well asother plant feedstuffs like Moringa (Moringa oleifera Lam.) leaves, allin various concentrations and combinations.

The protein sources may be in the form of non-treated plant materialsand treated and/or extracted plant proteins. As an example, heat treatedsoy products have high protein digestibility.

A protein material includes any type of protein or peptide. Inembodiments, soybean material or the like may be used such as wholesoybeans. Whole soybeans may be standard, commoditized soybeans;soybeans that have been genetically modified (GM) in some manner; ornon-GM identity preserved soybeans. Exemplary GM soybeans include, forexample, soybeans engineered to produce carbohydrates other thanstachyose and raffinose. Exemplary non-GM soybeans include, for example,Schillinger varieties that are line bred for low oil, low carbohydrates,and low trypsin inhibition.

Other types of soybean material include soy protein flour, soy proteinconcentrate, soybean meal and soy protein isolate, or mixtures thereof.The traditional processing of whole soybean into other forms of soyprotein such as soy protein flours, soy protein concentrates, soybeanmeal and soy protein isolates, includes cracking the cleaned, raw wholesoybean into several pieces, typically six(6) to eight (8), to producesoy chips and hulls, which are then removed. Soy chips are thenconditioned at about 60° C. and flaked to about 0.25 millimeterthickness. The resulting flakes are then extracted with an inertsolvent, such as a hydrocarbon solvent, typically hexane, in one ofseveral types of countercurrent extraction systems to remove the soybeanoil. For soy protein flours, soy protein concentrates, and soy proteinisolates, it is important that the flakes be desolventized in a mannerwhich minimizes the amount of cooking or toasting of the soy protein topreserve a high content of water-soluble soy protein. This is typicallyaccomplished by using vapour desolventizers or flash desolventizers. Theflakes resulting from this process are generally referred to as “edibledefatted flakes” or “white soy(bean) flakes.”

White soy bean flakes, which are the starting material for soy proteinflour, soy protein concentrate, and soy protein isolate, have a proteincontent of approximately 50%. White soybean flakes are then milled,usually in an open-loop grinding system, by a hammer mill, classifiermill, roller mill or impact pin mill first into grits, and withadditional grinding, into soy flours with desired particle sizes.Screening is typically used to size the product to uniform particle sizeranges, and can be accomplished with shaker screens or cylindricalcentrifugal screeners. Other oil seeds may be processed in a similarmanner.

In embodiments, distiller's dried grain solubles (DDGS) may be used.DDGS are currently manufactured by the corn ethanol industry.Traditional DDGS comes from dry grind facilities, in which the entirecorn kernel is ground and processed. DDGS in these facilities typicallycontains 28-32% protein.

The protein sources may be in the form of non-treated plant materialsand treated and/or extracted plant proteins. As an example, heat treatedsoy products have high protein digestibility. Still, the upper inclusionlevel for full fat or defatted soy meal inclusion in diets forcarnivorous fish is between an inclusion level of 20 to 30%, even ifheat labile antinutrients are eliminated. In fish, soybean protein hasshown that feeding fish with protein concentration inclusion levels over30% causes intestinal damage and in general reduces growth performancein different fish species. In fact, most fish farmers are reluctant touse more than 10% plant proteins in the total diet due to these effects.

The present invention solves this problem and allows for plant proteininclusion levels of up to 40 or even 50%, depending on, amongst otherfactors, the animal species being fed, the origin of the plant proteinsource, the ratio of different plant protein sources, the proteinconcentration and the amount, origin, molecular structure andconcentration of the glucan and/or mannan. In embodiments, the plantprotein inclusion levels are up to 40%, preferably up to 20 or 30%.Typically the plant protein present in the diet is between 5 and 40%,preferably between 10 or 15 and 30%. These percentages define thepercentage amount of a total plant protein source in the animal feed ordiet, this includes fat, ashes etc. In embodiments, pure protein levelsare up to 50%, typically up to 45%, in embodiments 5-95%.

The proportion of plant protein to other protein in the total feed ordiet may be 5:95 to 95:5, 15:85 to 50:50, or 25:75 to 45:55.

Microorganisms

The disclosed microorganisms must be capable of converting carbohydratesand other nutrients into a high-quality protein concentrate in aconversion culture. In embodiments, the microorganism is a yeast-likefungus. An example of a yeast-like fungus is Aurobasidium pullulans.Other example microorganisms include yeast such as Kluyveromyces andPichia spp, Lactic acid bacteria, Trichoderma reesei, Pleurotusostreatus, Rhizopus spp, and many types of lignocellulose degradingmicrobes. Generally, exemplary microbes include those microbes that canmetabolize stachyose, raffinose, xylose and other sugars. However, it iswithin the abilities of a skilled artisan to pick, without undueexperimentation, other appropriate microorganisms based on the disclosedmethods.

In embodiments, the microbial organisms that may be used in the presentprocess include, but are not limited to, Aureobasidium pullulans,Sclerotium glucanicum, Sphingomonas paucimobilis, Ralstonia eutropha,Rhodospirillum rubrum, Kluyveromyces and Pichia spp, Trichoderma reesei,Pleurotus ostreatus, Rhizopus spp, and combinations thereof. Inembodiments, the microbe is Aureobasidium pullulans.

In embodiments, the A. pullulans is adapted to variousenvironments/stressors encountered during conversion. In embodiments, anA. pullulans strain denoted by NRRL deposit No. 50793, which wasdeposited with the Agricultural Research Culture Collection (NRRL),Peoria, Ill., under the terms of the Budapest Treaty on Nov. 30, 2012,exhibits lower gum production and is adapted to DDGC. In embodiments, anA. pullulans strain denoted by NRRL deposit No. 50792, which wasdeposited with the Agricultural Research Culture Collection (NRRL),Peoria, Ill., under the terms of the Budapest Treaty on Nov. 30, 2012,is adapted to high levels of the antibiotic tetracycline (e.g., fromabout 75 μg/ml tetracycline to about 200 μg/ml tetracycline). Inembodiments, an A. pullulans strain denoted by NRRL deposit No. 50794,which was deposited with the Agricultural Research Culture Collection(NRRL), Peoria, Ill., under the terms of the Budapest Treaty on Nov. 30,2012, is adapted to high levels of the antibiotic LACTROL® (e.g., fromabout 2 μg/ml virginiamycin to about 6 μg/mlvirginiamycin). Inembodiments, an A. pullulans strain denoted by NRRL deposit No. 50795,which was deposited with the Agricultural Research Culture Collection(NRRL), Peoria, Ill., under the terms of the Budapest Treaty on Nov. 30,2012, is acclimated to condensed corn solubles.

Conversion Culture

In exemplary embodiments, after pretreatment, the protein material (suchas extruded soy white flakes) may be blended with water at a solidloading rate of at least 5%, with pH adjusted to 4.5-5.5. Thenappropriate dosages of hydrolytic enzymes may be added and the slurryincubated with agitation at 150-250 rpm at 50° C. for 3-24 h. Aftercooling to 35° C., an inoculum of A. pullulans may be added and theculture may be incubated for an additional 72-120 h, or until thecarbohydrates are consumed. During incubation, sterile air may besparged into the reactor at a rate of 0.5-1 L/L/h. In embodiments, theconversion culture undergoes conversion by incubation with the soybeanmaterial for less than about 96 hours. In embodiments, the conversionculture will be incubated for between about 96 hours and about 120hours. In embodiments, the conversion culture may be incubated for morethan about 120 hours. The conversion culture may be incubated at about35° C.

In embodiments, the pH of the conversion culture, while undergoingconversion, may be about 4.5 to about 5.5. In embodiments, the pH of theconversion culture may be less than 4.5 (e.g., at pH 3). In embodiments,the conversion culture may be actively aerated such as is disclosed inDeshpande et al., Aureobasidiumpullulans in applied microbiology: Astatus report, Enzyme and Microbial Technology (1992), 14(7):514.

The high-quality protein concentrate (HQPC), as well as pullulan andsiderophores, may be recovered from the conversion culture following theconversion process by optionally alcohol precipitation andcentrifugation. An example alcohol is ethanol, although the skilledartisan understands that other alcohols should work. In embodiments,salts may also be used to precipitate. Exemplary salts may be salts ofpotassium, sodium and magnesium chloride. In embodiments, a polymer ormutilvalent ions may be used alone or in combination with the alcohol.

In embodiments, final protein concentrations solids recovery may bemodulated by varying incubation times. For example, about 75% proteinmay be achieved with a 14 day incubation, where the solids recovery isabout 16-20%. In embodiments, incubation for 2-2.5 days increase solidsrecovery to about 60-64%, and protein level of 58-60% in the HQPC. Inembodiments, 4-5 day incubation may maximize both protein content (e.g.,but not limited to greater than about 70%) and solids recovery (e.g.,but not limited to greater than about 60%). These numbers may greater orlower, depending on the feed stock. In embodiments, the proteinconcentrates (i.e., HQSPC or HP-DDGS) may have a specific lipid:proteinratio, e.g., at about 0.010:1 to about 0.03:1, about 0.020:1 to about0.025:1 or about 0.021:1 to about 0.023:1.

In embodiments, feed stocks may be extruded in a single screw extruder(e.g., BRABENDER PLASTI-CORDER EXTRUDER Model PL2000, Hackensack, N.J.)with a barrel length to screw diameter of 1:20 and a compression ratioof 3:1, although other geometries and ratios may be used. Feed stocksmay be adjusted to about 10% to about 15% moisture, to about 15%, or toabout 25% moisture. The temperature of feed, barrel, and outlet sectionsof extruder may be held at between about 40° C. to about 50° C. or toabout 50° C. to about 100° C., about 100° C. to about 150° C., about150° C. to about 170° C., and screw speed may be set at about 50 rpm toabout 75 rpm or about 75 rpm to about 100 rpm or about 100 rpm to about200 rpm to about 250 rpm. In embodiments, the screw speed is sufficientto provide a shearing effect against the ridged channels on both sidesof a barrel. In embodiments, screw speed is selected to maximize sugarrelease.

In embodiments, extruded feed stock materials (e.g., plant proteins orDDGS) may be mixed with water to achieve a solid loading rate of atleast 5% in a reactor (e.g., a 5 L NEW BRUNSWICK BIOFLO 3 BIOREACTOR;3-4 L working volume). The slurry may be autoclaved, cooled, and thensaccharified by subjection to enzymatic hydrolysis using a cocktail ofenzymes including, but not limited to, endo-xylanase andbeta-xylosidase, Glycoside Hydrolase, ß-glucosidases, hemicellulaseactivities. In one aspect, the cocktail of enzymes includes NOVOZYME®enzymes. Dosages to be may include 6% CELLICCTEK® (per gm glucan), 0.3%CELLICHTEK® (per gm total solids), and 0.15% NOVOZYME 960® (per gm totalsolids). Saccharification may be conducted for about 12 h to about 24 hat 40° to about 50° C. and about 150 rpm to about 200 rpm to solubilizethe fibers and oligosaccharides into simple sugars. The temperature maythen be reduced to between about 30° C. to about 37° C., in embodimentsto about 35° C., and the slurry may be inoculated with 2% (v/v) of a 24h culture of the microbe. The slurry may be aerated at 0.5 L/L/min andincubation may be continued until sugar utilization ceases or about 96 hto about 120 h. In fed-batch conversions more extruded feed stock may beadded during either saccharification and/or the microbial conversionphase.

In embodiments, the feed stock and/or extrudate may be treated with oneor more antibiotics (e.g., but not limited to, tetracycline, penicillin,erythromycin, tylosin, virginiamycin, and combinations thereof) beforeinoculation with the converting microbe to avoid, for example,contamination by unwanted bacteria strains.

During incubation, samples may be removed at 6-12 h intervals. Samplesfor HPLC analysis may be boiled, centrifuged, filtered (e.g., through0.22-μm filters), placed into autosampler vials, and frozen untilanalysis. In embodiments, samples may be assayed for carbohydrates andorganic solvents using a WATERS HPLC system, although other HPLC systemsmay be used. Samples may be subjected to plate or hemocytometer countsto assess microbial populations. Samples may also be assayed for levelsof cellulose, hemicellulose, and pectin using National Renewable EnergyLaboratory procedures.

Dietary Formulations

In exemplary embodiments, the high-quality protein concentrate recoveredfrom the conversion culture that has undergone conversion is used indietary formulations. In embodiments, the recovered high-quality proteinconcentrate (HQPC) will be the primary protein source in the dietaryformulation. Protein source percentages in dietary formulations are notmeant to be limiting and may include 24 to 80% protein. In embodiments,the high-quality protein concentrate (HQPC) will be more than about 50%,more than about 60%, or more than about 70% of the total dietaryformulation protein source. Recovered HQPC may replace protein sourcessuch as fish meal, soybean meal, wheat and corn flours and glutens andconcentrates, and animal byproduct such as blood, poultry, and feathermeals. Dietary formulations using recovered HQPC may also includesupplements such as mineral and vitamin premixes to satisfy remainingnutrient requirements as appropriate.

In certain embodiments, performance of the HQPC, such as high-qualitysoy protein concentrate (HQSPC) or high-quality DDGS (HP-DDGS), may bemeasured by comparing the growth, feed conversion, protein efficiency,and survival of animal on a high-quality protein concentrate dietaryformulation to animals fed control dietary formulations, such asfish-meal. In embodiments, test formulations contain consistent protein,lipid, and energy contents. For example, when the animal is a fish,viscera (fat deposition) and organ (liver and spleen) characteristics,dress-out percentage, and fillet proximate analysis, as well asintestinal histology (enteritis) may be measured to assess dietaryresponse.

As is understood, individual dietary formulations containing therecovered HQPC may be optimized for different kinds of animals. Inembodiments, the animals are fish grown in commercial aquaculture.Methods for optimization of dietary formulations are well-known andeasily ascertainable by the skilled artisan without undueexperimentation.

Complete grower diets may be formulated using HQPC in accordance withknown nutrient requirements for various animal species. In embodiments,the formulation may be used for yellow perch (e.g., 42% protein, 8%lipid). In embodiments, the formulation may be used for rainbow trout(35% protein, 16% lipid). In embodiments, the formulation may be usedfor any one of the animals recited supra.

Basal mineral and vitamin premixes for plant-based diets may be used toensure that micro-nutrient requirements will be met. Any supplements (asdeemed necessary by analysis) may be evaluated by comparing to anidentical formulation without supplementation; thus, the feeding trialmay be done in a factorial design to account for supplementationeffects. In embodiments, feeding trials may include a fish meal-basedcontrol diet and ESPC- and LSPC-based reference diets [traditional SPC(TSPC) is produced from solvent washing soy flake to remove solublecarbohydrate; texturized SPC (ESPC) is produced by extruding TSPC undermoist, high temperature; and low-antigen SPC (LSPC) is produced fromTSPC by altering the solvent wash and temperature during processing].Pellets for feeding trials may be produced using the lab-scale singlescrew extruder (e.g., BRABENDERPLASTI-CORDER EXTRUDER Model PL2000).

Feeding Trials

In embodiments, a replication of four experimental units per treatment(i.e., each experimental and control diet blend) may be used (e.g.,about 60 to 120 days each). Trials may be carried out in 110-L circulartanks (20 fish/tank) connected in parallel to a closed-looprecirculation system driven by a centrifugal pump and consisting of asolids sump, and bioreactor, filters (100 μm bag, carbon andultra-violet). Heat pumps may be used as required to maintain optimaltemperatures for species-specific growth. Water quality (e.g., dissolvedoxygen, pH, temperature, ammonia and nitrite) may be monitored in allsystems.

In embodiments, experimental diets may be delivered according to fishsize and split into two to five daily feedings. Growth performance maybe determined by total mass measurements taken at one to four weeks(depending upon fish size and trial duration); rations may be adjustedin accordance with gains to allow satiation feeding and to reduce wastestreams. Consumption may be assessed biweekly from collections ofuneaten feed from individual tanks. Uneaten feed may be dried to aconstant temperature, cooled, and weighed to estimate feed conversionefficiency. Protein and energy digestibilities may determined from fecalmaterial manually stripped during the midpoint of each experiment or vianecropsy from the lower intestinal tract at the conclusion of a feedingtrial. Survival, weight gain, growth rate, health indices, feedconversion, protein and energy digestibilities, and protein efficiencymay be compared among treatment groups. Proximate analysis of necropsiedfishes may be carried out to compare composition of fillets amongdietary treatments. Analysis of amino and fatty acids may be done asneeded for fillet constituents, according to the feeding trialobjective. Feeding trial responses of dietary treatments may be comparedto a control (e.g., fish meal) diet response to ascertain whetherperformance of HQPC diets meet or exceed control responses.

Statistical analyses of diets and feeding trial responses may becompleted with an a priori α=0.05. Analysis of performance parametersamong treatments may be performed with appropriate analysis of varianceor covariance (Proc Mixed) and post hoc multiple comparisons, as needed.Analysis of fish performance and tissue responses may be assessed bynonlinear models.

In embodiments, the present disclosure proposes to convert fibers andother carbohydrates in soy flakes/meal or DDGS into additional proteinusing, for example, a GRAS-status microbe. A microbial exopolysaccharide(i.e., gum) may also be produced that may facilitate extruded feedpellet formation, negating the need for binders. This microbial gum mayalso provide immunostimulant activity to activate innate defensemechanisms that protect fish from common pathogens resulting fromstressors. Immunoprophylactic substances, such as (3-glucans, bacterialproducts, and plant constituents, are increasingly used in commercialfeeds to reduce economic losses due to infectious diseases and minimizeantibiotic use. The microbes of the present disclosure also produceextracellular peptidases, which should increase corn proteindigestibility and absorption during metabolism, providing higher feedefficiency and yields. As disclosed herein, this microbial incubationprocess provides a valuable, sustainable aquaculture feed that is lessexpensive per unit of protein than SBM, SPC, and fish meal.

As disclosed, the instant microbes may metabolize the individualcarbohydrates in soy flakes/meal or DDGS, producing both cell mass(protein) and a microbial gum. Various strains of these microbes alsoenhance fiber deconstruction. The microbes of the present invention mayalso convert soy and corn proteins into more digestible peptides andamino acids. In embodiments, the following actions in may beperformed: 1) determining the efficiency of using select microbes of thepresent disclosure to convert pretreated soy protein, oil seed proteins,DDGS and the like, yielding a high quality protein concentrate (HQPC)with a protein concentration of at least 45%, and 2) assessing theeffectiveness of HQPC in replacing fish meal. In embodiments, optimizingsoy, oil seed, and DDGS pretreatment and conversion conditions may becarried out to improve the performance and robustness of the microbes,test the resultant grower feeds for a range of commercially importantfishes, validate process costs and energy requirements, and completesteps for scale-up and commercialization. In embodiments, the HQPC ofthe present disclosure may be able to replace at least 50% of fish meal,while providing increased growth rates and conversion efficiencies.Production costs should be less than commercial soy protein concentrate(SPC) and substantially less than fish meal (including harvest).

FIGS. 1 and 2 show the approach of the present disclosure in thepretreatment of plant based product, converting sugars into cell mass(protein) and gum, recovering HQSPC and generating aqua feeds, andtesting the resulting aqua feeds in fish feeding trials.

After extrusion pretreatment, cellulose-deconstructing enzymes may beevaluated to generate sugars, which microbes of the present disclosuremay convert to protein and gum. In embodiments, sequential omission ofthese enzymes and evaluation of co-culturing with cellulolytic microbesmay be used. Ethanol may be evaluated to precipitate the gum and improvecentrifugal recovery of the HQPC. After drying, the HQPC may beincorporated into practical diet formulations. In embodiments, testgrower diets may be formulated (with mineral and vitamin premixes) andcomparisons to a fish-meal control and commercial SPC (SPC is distinctlydifferent from soybean meal, as it contains traces ofoligopolysaccharides and antigenic substances glycinin andb-conglycinin) diets in feeding trials with a commercially importantfish, e.g., yellow perch or rainbow trout, may be performed. Performance(e.g., growth, feed conversion, protein efficiency), visceracharacteristics, and intestinal histology may be examined to assess fishresponses.

In other embodiments, optimizing the HQPC production process bydetermining optimum pretreatment and conversion conditions whileminimizing process inputs, improving the performance and robustness ofthe microbe, testing the resultant grower feeds for a range ofcommercially important fishes, validating process costs and energyrequirements, and completing initial steps for scale-up andcommercialization may be carried out.

In the past few years, a handful of facilities have installed a dry millcapability that removes corn hulls and germ prior to the ethanolproduction process. This dry fractionation process yields a DDGS with upto 42% protein (hereafter referred to as dryfrac DDGS). In embodiments,conventional and dryfrac DDGS under conditions previously determined torapidly generate a sufficient amount of high protein DDGS (HP-DDGS) foruse in perch feeding trials may be compared. In embodiments, carefulmonitoring of the performance of this conversion (via chemicalcomposition changes) is carried out and parameters with the greatestimpact on HP-DDGS quality identified. In some embodiments, low oil DDGSmay be used as a substrate for conversion, where such low oil DDGS has ahigher protein level than conventional DDGS. In a related aspect, lowoil DDGS increase growth rates of A. pullulans compared to conventionalDDGS.

Several groups are evaluating partial replacement of fish-meal withplant derived proteins, such as soybean meal and DDGS. However, thelower protein content, inadequate amino acid balance, and presence ofanti-nutritional factors have limited the replacement levels to 20-40%.Preliminary growth trials indicate that no current DDGS or SPC-baseddiets provide performance similar to fish-meal control diets. Severaldeficiencies have been identified among commercially produced DDGS andSPCs, principally in protein and amino acid composition, which impartvariability in growth performance and fish composition. However, HP-DDGSand HQSPC diets as disclosed herein containing nutritional supplements(formulated to meet or exceed all requirements) have provided growthresults that are similar to or exceed fish-meal controls. Thus, theprocesses as disclosed herein and products developed therefrom provide ahigher quality HQSPC or HP-DDGS (relative to nutritional requirements)and support growth performance equivalent to or better than dietscontaining fish meal.

Fish that can be fed the fish feed composition of the present disclosureinclude, but are not limited to, Siberian sturgeon, Sterlet sturgeon,Starry sturgeon, White sturgeon, Arapaima, Japanese eel, American eel,Short-finned eel, Long-finned eel, European eel, Chanos chanos,Milkfish, Bluegill sunfish, Green sunfish, White crappie, Black crappie,Asp, Catla, Goldfish, Crucian carp, Mud carp, Mrigal carp, Grass carp,Common carp, Silver carp, Bighead carp, Orangefin labeo, Roho labeo,Hoven's carp, Wuchang bream, Black carp, Golden shiner, Nilem carp,White amur bream, Thai silver barb, Java, Roach, Tench, Pond loach,Bocachico, Dorada, Cachama, Cachama Blanca, Paco, Black bullhead,Channel catfish, Bagrid catfish, Blue catfish, Wels catfish, Pangasius(Swai, Tra, Basa) catfish, Striped catfish, Mudfish, Philippine catfish,Hong Kong catfish, North African catfish, Bighead catfish, Sampa, SouthAmerican catfish, Atipa, Northern pike, Ayu sweetfish, Vendace,Whitefish, Pink salmon, Chum salmon, Coho salmon, Masu salmon, Rainbowtrout, Sockeye salmon, Chinook salmon, Atlantic salmon, Sea trout,Arctic char, Brook trout, Lake trout, Atlantic cod, Pejerrey, Lai,Common snook, Barramundi/Asian sea bass, Nile perch, Murray cod, Goldenperch, Striped bass, White bass, European seabass, Hong Kong grouper,Areolate grouper, Greasy grouper, Spotted coralgrouper, Silver perch,White perch, Jade perch, Largemouth bass, Smallmouth bass, Europeanperch, Zander (Pike-perch), Yellow Perch, Sauger, Walleye, Bluefish,Greater amberjack, Japanese amberjack, Snubnose pompano, Floridapompano, Palometa pompano, Japanese jack mackerel, Cobia, Mangrove redsnapper, Yellowtail snapper, Dark seabream, White seabream, Crimsonseabream, Red seabream, Red porgy, Goldlined seabream, Giltheadseabream, Red drum, Green terror, Blackbelt cichlid, Jaguar guapote,Mexican mojarra, Pearlspot, Three spotted tilapia, Blue tilapia, Longfintilapia, Mozambique tilapia, Nile tilapia, Tilapia, Wami tilapia,Blackchin tilapia, Redbreast tilapia, Redbelly tilapia, Golden greymullet, Largescale mullet, Gold-spot mullet, Thinlip grey mullet,Leaping mullet, Tade mullet, Flathead grey mullet, White mullet,Lebranche mullet, Pacific fat sleeper, Marble goby, White-spottedspinefoot, Goldlined spinefoot, Marbled spinefoot, Southern bluefintuna, Northern bluefin tuna, Climbing perch, Snakeskin gourami, Kissinggourami, Giant gourami, Snakehead, Indonesian snakehead, Spottedsnakehead, Striped snakehead, Turbot, Bastard halibut (Japaneseflounder), Summer Flounder, Southern flounder, Winter flounder, AtlanticHalibut, Greenback flounder, Common sole, and combinations thereof

It will be appreciated by the skilled person that the fish feedcomposition of the present disclosure may be used as a convenientcarrier for pharmaceutically active substances such as for exampleantimicrobial agents and immunologically active substances includingvaccines against bacterial or viral infections, and any combinationthereof.

The fish feed composition according to present disclosure may beprovided as a liquid, pourable emulsion, or in the form of a paste, orin a dry form, for example as a granulate or pellet, a powder, or asflakes. When the fish feed composition is provided as an emulsion, alipid-in-water emulsion, it is may be in a relatively concentrated form.Such a concentrated emulsion form may also be referred to as apre-emulsion as it may be diluted in one or more steps in an aqueousmedium to provide the final enrichment medium for the organisms.

In embodiments, cellulosic-containing starting material for themicrobial-based process as disclosed is corn. Corn is about two-thirdsstarch, which is converted during a fermentation and distilling processinto ethanol and carbon dioxide. The remaining nutrients or fermentationproducts may result in condensed distiller's solubles or distiller'sgrains such as DDGS, which can be used in feed products. In general, theprocess involves an initial preparation step of dry milling or grindingof the corn. The processed corn is then subject to hydrolysis andenzymes added to break down the principal starch component in asaccharification step. The following step of fermentation is allowed toproceed upon addition of a microorganism (e.g., yeast) provided inaccordance with an embodiment of the disclosure to produce gaseousproducts such as carbon dioxide. The fermentation is conducted for theproduction of ethanol which can be distilled from the fermentationbroth. The remainder of the fermentation medium can be then dried toproduce fermentation products including DDGS. This step usually includesa solid/liquid separation process by centrifugation wherein a solidphase component can be collected. Other methods including filtration andspray dry techniques can be employed to effect such separation. Theliquid phase components can be subjected further afterwards to anevaporation step that can concentrate soluble coproducts, such assugars, glycerol and amino acids, into a material called syrup orcondensed corn solubles (CCS). The CCS can then be recombined with thesolid phase component to be dried as incubation products (DDGS). Itshall be understood that the subject compositions and can be applied tonew or already existing ethanol plants based on dry milling to providean integrated ethanol production process that also generatesfermentation products with increased value.

In embodiments, incubation products produced according to the presentdisclosure have a higher commercial value than the conventionalfermentation products. For example, the incubation products may includeenhanced dried solids with improved amino acid and micronutrientcontent. A “golden colored” product can be thus provided which generallyindicates higher amino acid digestibility compared to darker coloredHQSP. For example, a light-colored HQSP may be produced with anincreased lysine concentration in accordance with embodiments hereincompared to relatively darker colored products with generally lessnutritional value. The color of the products may be an important factoror indicator in the assessing the quality and nutrient digestibility ofthe fermentation products or HQSP. Color is used as an indicator ofexposure to excess heat during drying causing caramelization and Millardreactions of the free amino groups and sugars, reducing the quality ofsome amino acids.

The basic steps in a dry mill or grind ethanol manufacturing process maybe described as follows: milling or grinding of corn or other grainproduct, saccharification, fermentation, and distillation. For example,selected whole corn kernels may be milled or ground with typicallyeither hammer mills or roller mills. The particle size can influencecooking hydration and subsequent enzymatic conversion. The milled orground corn can be then mixed with water to make a mash that is cookedand cooled. It may be useful to include enzymes during the initial stepsof this conversion to decrease the viscosity of the gelatinized starch.The mixture may be then transferred to saccharification reactors,maintained at selected temperatures such as 140° F., where the starch isconverted by addition of saccharifying enzymes to fermentable sugarssuch as glucose or maltose. The converted mash can be cooled to desiredtemperatures such as 84° F., and fed to fermentation reactors wherefermentable sugars are converted to carbon dioxide by the use ofselected strains of microbes provided in accordance with the disclosurethat results in more nutritional fermentation products compared to moretraditional ingredients such as Saccharomyces yeasts. The resultingproduct can be flashed to separate out carbon dioxide and the resultingliquid can be fed to a recovery system consisting of distillationcolumns and a stripping column. The ethanol stream can be directed to amolecular sieve where remaining water is removed using adsorptiontechnology. Purified ethanol, denatured with a small amount of gasoline,can produce fuel grade ethanol. Another product can be produced byfurther purifying the initial distillate ethanol to remove impurities,resulting in about 99.95% ethanol for non-fuel uses.

The whole stillage can be withdrawn from the bottom of the distillationunit and centrifuged to produce distiller's wet grains (DWG) and thinstillage (liquids). The DWG can leave the centrifuge at 55-65% moisture,and can either be sold wet as a cattle feed or dried as enhancedfermentation products provided in accordance with the disclosure. Theseproducts include an enhanced end product that may be referred to hereinas distiller's dried grains (DDG). Using an evaporator, the thinstillage (liquid) can be concentrated to form distiller's solubles,which can be added back to and combined with a distiller's grainsprocess stream and dried. This combined product in accordance withembodiments of the disclosure may be marketed as an enhancedfermentation product having increased amino acid and micronutrientcontent. It shall be understood that various concepts of the disclosuremay be applied to other fermentation processes known in the field otherthan those illustrated herein.

Another aspect of the present invention is directed towards completefish meal compositions with an enhanced concentration of nutrients whichincludes microorganisms characterized by an enhanced concentration ofnutrients such as, but not limited to, fats, fatty acids, lipids such asphospholipid, vitamins, essential amino acids, peptides, proteins,carbohydrates, sterols, enzymes, and trace minerals such as, iron,copper, zinc, manganese, cobalt, iodine, selenium, molybdenum, nickel,fluorine, vanadium, tin, silicon, and combinations thereof.

In a incubation process of the present disclosure, a carbon source maybe hydrolyzed to its component sugars by microorganisms to producealcohol and other gaseous products. Gaseous product includes carbondioxide and alcohol includes ethanol. The incubation products obtainedafter the incubation process are typically of higher commercial value.In embodiments, the incubation products contain microorganisms that haveenhanced nutrient content than those products deficient in themicroorganisms. The microorganisms may be present in an incubationsystem, the incubation broth and/or incubation biomass. The incubationbroth and/or biomass may be dried (e.g., spray-dried), to produce theincubation products with an enhanced content of the nutritionalcontents.

For example, the spent, dried solids recovered following the incubationprocess are enhanced in accordance with the disclosure. These incubationproducts are generally non-toxic, biodegradable, readily available,inexpensive, and rich in nutrients. The choice of microorganism and theincubation conditions are important to produce a low toxicity ornon-toxic incubation product for use as a feed or nutritionalsupplement. While glucose is the major sugar produced from thehydrolysis of the starch from grains, it is not the only sugar producedin carbohydrates generally. Unlike the SPC or DDG produced from thetraditional dry mill ethanol production process, which contains a largeamount of non-starch carbohydrates (e.g., as much as 35% percent ofcellulose and arabinoxylans-measured as neutral detergent fiber, by dryweight), the subject nutrient enriched incubation products produced byenzymatic hydrolysis of the non-starch carbohydrates are more palatableand digestible to the non-ruminant.

The nutrient enriched incubation product of this disclosure may have anutrient content of from at least about 1% to about 95% by weight. Thenutrient content is preferably in the range of at least about 10%-20%,20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, and 70%-80% by weight. Theavailable nutrient content may depend upon the animal to which it is fedand the context of the remainder of the diet, and stage in the animallife cycle. For instance, beef cattle require less histidine thanlactating cows. Selection of suitable nutrient content for feedinganimals is well known to those skilled in the art.

The incubation products may be prepared as a spray-dried biomassproduct. Optionally, the biomass may be separated by known methods, suchas centrifugation, filtration, separation, decanting, a combination ofseparation and decanting, ultrafiltration or microfiltration. Thebiomass incubation products may be further treated to facilitate rumenbypass. In embodiments, the biomass product may be separated from theincubation medium, spray-dried, and optionally treated to modulate rumenbypass, and added to feed as a nutritional source. In addition toproducing nutritionally enriched incubation products in a incubationprocess containing microorganisms, the nutritionally enriched incubationproducts may also be produced in transgenic plant systems. Methods forproducing transgenic plant systems are known in the art. Alternatively,where the microorganism host excretes the nutritional contents, thenutritionally-enriched broth may be separated from the biomass producedby the incubation and the clarified broth may be used as an animal feedingredient, e.g., either in liquid form or in spray dried form.

The incubation products obtained after the incubation process usingmicroorganisms may be used as an animal feed or as food supplement forhumans. The incubation product includes at least one ingredient that hasan enhanced nutritional content that is derived from a non-animal source(e.g., a bacteria, yeast, and/or plant). In particular, the incubationproducts are rich in at least one or more of fats, fatty acids, lipidssuch as phospholipid, vitamins, essential amino acids, peptides,proteins, carbohydrates, sterols, enzymes, and trace minerals such as,iron, copper, zinc, manganese, cobalt, iodine, selenium, molybdenum,nickel, fluorine, vanadium, tin and silicon. In embodiments, thepeptides contain at least one essential amino acid. In otherembodiments, the essential amino acids are encapsulated inside a subjectmodified microorganism used in an incubation reaction. In embodiments,the essential amino acids are contained in heterologous polypeptidesexpressed by the microorganism. Where desired, the heterologouspolypeptides are expressed and stored in the inclusion bodies in asuitable microorganism (e.g., fungi).

In embodiments, the incubation products have a high nutritional content.As a result, a higher percentage of the incubation products may be usedin a complete animal feed. In embodiments, the feed compositioncomprises at least about 15% of incubation product by weight. In acomplete feed, or diet, this material will be fed with other materials.Depending upon the nutritional content of the other materials, and/orthe nutritional requirements of the animal to which the feed isprovided, the modified incubation products may range from 15% of thefeed to 100% of the feed. In embodiments, the subject incubationproducts may provide lower percentage blending due to high nutrientcontent. In other embodiments, the subject incubation products mayprovide very high fraction feeding, e.g. over 75%. In suitableembodiments, the feed composition comprises at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 60%, at leastabout 70%, or at least about 75% of the subject incubation products.Commonly, the feed composition comprises at least about 20% ofincubation product by weight. More commonly, the feed compositioncomprises at least about 15-25%, 25-20%, 20-25%, 30%-40%, 40%-50%,50%-60%, or 60%-70% by weight of incubation product. Where desired, thesubject incubation products may be used as a sole source of feed.

The complete fish meal compositions may have enhanced amino acid contentwith regard to one or more essential amino acids for a variety ofpurposes, e.g., for weight increase and overall improvement of theanimal's health. The complete fish meal compositions may have anenhanced amino acid content because of the presence of free amino acidsand/or the presence of proteins or peptides including an essential aminoacid, in the incubation products. Essential amino acids may includearginine, cysteine, histidine, isoleucine, lysine, methionine,phenylalanine, threonine, taurine, tryptophan, and/or valine, which maybe present in the complete animal feed as a free amino acid or as partof a protein or peptide that is rich in the selected amino acid. Atleast one essential amino acid-rich peptide or protein may have at least1% essential amino acid residues per total amino acid residues in thepeptide or protein, at least 5% essential amino acid residues per totalamino acid residues in the peptide or protein, or at least 10% essentialamino acid residues per total amino acid residues in the protein. Byfeeding a diet balanced in nutrients to animals, maximum use is made ofthe nutritional content, requiring less feed to achieve comparable ratesof growth, milk production, or a reduction in the nutrients present inthe excreta reducing bioburden of the wastes.

A complete fish meal composition with an enhanced content of anessential amino acid, may have an essential amino acid content(including free essential amino acid and essential amino acid present ina protein or peptide) of at least 2.0 wt % relative to the weight of thecrude protein and total amino acid content, and more suitably at least5.0 wt % relative to the weight of the crude protein and total aminoacid content. The complete fish meal composition includes othernutrients derived from microorganisms including but not limited to,fats, fatty acids, lipids such as phospholipid, vitamins, carbohydrates,sterols, enzymes, and trace minerals.

The complete fish meal composition may include complete feed formcomposition, concentrate form composition, blender form composition, andbase form composition. If the composition is in the form of a completefeed, the percent nutrient level, where the nutrients are obtained fromthe microorganism in an incubation product, which may be about 10 toabout 25 percent, more suitably about 14 to about 24 percent; whereas,if the composition is in the form of a concentrate, the nutrient levelmay be about 30 to about 50 percent, more suitably about 32 to about 48percent. If the composition is in the form of a blender, the nutrientlevel in the composition may be about 20 to about 30 percent, moresuitably about 24 to about 26 percent; and if the composition is in theform of a base mix, the nutrient level in the composition may be about55 to about 65 percent. Unless otherwise stated herein, percentages arestated on a weight percent basis. If the HQPC is high in a singlenutrient, e.g., Lys, it will be used as a supplement at a low rate; ifit is balanced in amino acids and Vitamins, e.g., vitamin A and E, itwill be a more complete feed and will be fed at a higher rate andsupplemented with a low protein, low nutrient feed stock, like cornstover.

The fish meal composition may include a peptide or a crude proteinfraction present in an incubation product having an essential amino acidcontent of at least about 2%. In embodiments, a peptide or crude proteinfraction may have an essential amino acid content of at least about 3%,at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 30%, at least about 40%, and in embodiments,at least about 50%. In embodiments, the peptide may be 100% essentialamino acids. Commonly, the fish meal composition may include a peptideor crude protein fraction present in an incubation product having anessential amino acid content of up to about 10%. More commonly, the fishmeal composition may include a peptide or a crude protein fractionpresent in an incubation product having an essential amino acid contentof about 2-10%, 3.0-8.0%, or 4.0-6.0%.

The fish meal composition may include a peptide or a crude proteinfraction present in a incubation product having a lysine content of atleast about 2%. In embodiments, the peptide or crude protein fractionmay have a lysine content of at least about 3%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about30%, at least about 40%, and in embodiments, at least about 50%.Typically, the fish meal composition may include the peptide or crudeprotein fraction having a lysine content of up to about 10%. Wheredesired, the fish meal composition may include the peptide or a crudeprotein fraction having a lysine content of about 2-10%, 3.0-8.0%, or4.0-6.0%.

The fish meal composition may include nutrients in the incubationproduct from about 1 g/Kg dry solids to 900 g/Kg dry solids. Inembodiments, the nutrients in a fish meal composition may be present toat least about 2 g/Kg dry solids, 5 g/Kg dry solids, 10 g/Kg dry solids,50 g/Kg dry solids, 100 g/Kg dry solids, 200 g/Kg dry solids, and about300 g/Kg dry solids. In embodiments, the nutrients may be present to atleast about 400 g/Kg dry solids, at least about 500 g/Kg dry solids, atleast about 600 g/Kg dry solids, at least about 700 g/Kg dry solids, atleast about 800 g/Kg dry solids and/or at least about 900 g/Kg drysolids.

The fish meal composition may include an essential amino acid or apeptide containing at least one essential amino acid present in anincubation product having a content of about 1 g/Kg dry solids to 900g/Kg dry solids. In embodiments, the essential amino acid or a peptidecontaining at least one essential amino acid in a fish meal compositionmay be present to at least about 2 g/Kg dry solids, 5 g/Kg dry solids,10 g/Kg dry solids, 50 g/Kg dry solids, 100 g/Kg dry solids, 200 g/Kgdry solids, and about 300 g/Kg dry solids. In embodiments, the essentialamino acid or a peptide containing at least one essential amino acid maybe present to at least about 400 g/Kg dry solids, at least about 500g/Kg dry solids, at least about 600 g/Kg dry solids, at least about 700g/Kg dry solids, at least about 800 g/Kg dry solids and/or at leastabout 900 g/Kg dry solids.

The complete fish meal composition may contain a nutrient enrichedincubation product in the form of a biomass formed during incubation andat least one additional nutrient component. In another example, the fishmeal composition contains a nutrient enriched incubation product that isdissolved and suspended from an incubation broth formed duringincubation and at least one additional nutrient component. In a furtherembodiment, the fish meal composition has a crude protein fraction thatincludes at least one essential amino acid-rich protein. The fish mealcomposition may be formulated to deliver an improved balance ofessential amino acids.

For compositions comprising DDGS, the complete composition form maycontain one or more ingredients such as wheat middlings (“wheat mids”),corn, soybean meal, corn gluten meal, distiller's grains or distiller'sgrains with solubles, salt, macro-minerals, trace minerals and vitamins.Other potential ingredients may commonly include, but not be limited tosunflower meal, malt sprouts and soybean hulls. The blender formcomposition may contain wheat middlings, corn gluten meal, distiller'sgrains or distiller's grains with solubles, salt, macro-minerals, traceminerals and vitamins. Alternative ingredients would commonly include,but not be limited to, corn, soybean meal, sunflower meal, cottonseedmeal, malt sprouts and soybean hulls. The base form composition maycontain wheat middlings, corn gluten meal, and distiller's grains ordistiller's grains with solubles. Alternative ingredients would commonlyinclude, but are not limited to, soybean meal, sunflower meal, maltsprouts, macro-minerals, trace minerals and vitamins.

Highly unsaturated fatty acids (HUFAs) in microorganisms, when exposedto oxidizing conditions may be converted to less desirable unsaturatedfatty acids or to saturated fatty acids. However, saturation of omega-3HUFAs may be reduced or prevented by the introduction of syntheticantioxidants or naturally-occurring antioxidants, such as beta-carotene,vitamin E and vitamin C, into the feed. Synthetic antioxidants, such asBHT, BHA, TBHQ or ethoxyquin, or natural antioxidants such astocopherols, may be incorporated into the food or feed products byadding them to the products, or they may be incorporated by in situproduction in a suitable organism. The amount of antioxidantsincorporated in this manner depends, for example, on subsequent userequirements, such as product formulation, packaging methods, anddesired shelf life.

Incubation products or complete fish meal containing the incubationproducts of the present disclosure, may also be utilized as anutritional supplement for human consumption if the process begins withhuman grade input materials, and human food quality standards areobserved through out the process. Incubation product or the completefeed as disclosed herein is high in nutritional content. Nutrients suchas, protein and fiber are associated with healthy diets. Recipes may bedeveloped to utilize incubation product or the complete feed of thedisclosure in foods such as cereal, crackers, pies, cookies, cakes,pizza crust, summer sausage, meat balls, shakes, and in any forms ofedible food. Another choice may be to develop the incubation product orthe complete feed of the disclosure into snacks or a snack bar, similarto a granola bar that could be easily eaten, convenient to distribute. Asnack bar may include protein, fiber, germ, vitamins, minerals, from thegrain, as well as nutraceuticals such as glucosamine, HUFAs, orco-factors, such as Vitamin Q-10.

The fish meal comprising the subject incubation products may be furthersupplemented with flavors. The choice of a particular flavor will dependon the animal to which the feed is provided. The flavors and aromas,both natural and artificial, may be used in making feeds more acceptableand palatable. These supplementations may blend well with allingredients and may be available as a liquid or dry product form.Suitable flavors, attractants, and aromas to be supplemented in theanimal feeds include but not limited to fish pheromones, fenugreek,banana, cherry, rosemary, cumin, carrot, peppermint oregano, vanilla,anise, plus rum, maple, caramel, citrus oils, ethyl butyrate, menthol,apple, cinnamon, any natural or artificial combinations thereof. Thefavors and aromas may be interchanged between different animals.Similarly, a variety of fruit flavors, artificial or natural may beadded to food supplements comprising the subject incubation products forhuman consumption.

The shelf-life of the incubation product or the complete feed of thepresent disclosure may typically be longer than the shelf life of anincubation product that is deficient in the microorganism. Theshelf-life may depend on factors such as, the moisture content of theproduct, how much air can flow through the feed mass, the environmentalconditions and the use of preservatives. A preservative may be added tothe complete feed to increase the shelf life to weeks and months. Othermethods to increase shelf life include management similar to silagemanagement such as mixing with other feeds and packing, covering withplastic or bagging. Cool conditions, preservatives and excluding airfrom the feed mass all extend shelf life of wet co-products. Thecomplete feed can be stored in bunkers or silo bags. Drying the wetincubation product or complete feed may also increase the product'sshelf life and improve consistency and quality.

The complete feed of the present disclosure may be stored for longperiods of time. The shelf life may be extended by ensiling, addingpreservatives such as organic acids, or blending with other feeds suchas soy hulls. Commodity bins or bulk storage sheds may be used forstoring the complete feeds.

As used herein, “room temperature” is about 25° C. under standardpressure.

The following examples are illustrative and are not intended to limitthe scope of the disclosed subject matter.

EXAMPLES Example 1. High Quality Soy Protein Concentrate (HQSPC)

FIG. 1 shows the overall approach to pre-treating white flakes,converting sugars into cell mass (protein) and gum, recovering HQSPC andgenerating aquafeeds (FIG. 2), and testing resulting aquafeeds in fishfeeding trials. White flakes were first subject to extrusionpretreatment (BRABENDER PLASTI-CORDER SINGLE SCREW EXTRUDER ModelPL2000, Hackensack, N.J.) at 15% moisture content, 50° C., and 75 rpm todisrupt the structure and allow increased intrusion of hydrolyticenzymes during subsequent saccharification. These conditions provided ashearing effect against the rigged channels on both sides of the barrel,and it had been observed previously that this resulted in 50-70% greatersugar release following enzymatic hydrolysis. Extruded white flakes werethen ground through a 3 mm hammermill screen, blended with water toachieve a 10% solid loading rate, and adjusted to pH 5. After heating topasteurize or sterilize the mash, the mash was cooled to about 50° C.and cellulose and oligosaccharide-deconstructing enzymes (15 ml total/kgof white flake) were added to hydrolyze the polymers into simple sugars(4-24 h hydrolysis). Specific dosages included were 6% CELLIC CTEK (pergm glucan), 0.3% CELLIC HTEK (per gm total solids), 0.015% NOVOZYME 960(per gm solids). The resulting mash was then cooled to 30° C., pHadjusted to 3-5, inoculated with A. pullulans (1% v/v), and incubatedfor 4-5 days at 50 to 200 rpm mixing and an aeration rate of 0.5 L/L/minto convert sugars into protein and gum. During incubation, samples wereperiodically removed and analyzed for sugars, cell counts and gumproduction. Following incubation, the pH was increased to 6.5, andethanol (0.6 L/L broth) was added to precipitate the gum. The protein,pullulan and microbial mass (HQSPC) were recovered by centrifugation anddried, while the supernatant was distilled to recover ethanol, and theresidual liquid chemically assayed for future recycling at the start ofthe process. The HQSPC was then tested in feeding trials with yellowperch, a fish of regional market importance. Test grower diets wereformulated with HQ SPC compared to fishmeal and a competing plant basedingredient. Performance (e.g., growth, feed conversion, proteinefficiency), viscera characteristics, and intestinal histology wereexamined to assess fish responses.

HQSPC using Soy White Flake and Microbial Conversion with A. Pullulans,pilot scale system for the production of HQSPC.

The system contained a 675 L bioreactor, a variable speed progressivecavity pump, a continuous flow centrifuge, and a 1×4 meter drying table.Inoculum for use in the 675 L bioreactor was prepared in two, 5 L NEWBRUNSWICK BIOFLO 3 BIOREACTORS. For each trial 8-10 L quantities ofinoculum was prepared by growing A. pullulan as described for 2-3 days.This material was used to inoculate larger quantities of extruded andsaccharified white flake prepared in the 675 L bioreactor. Followingincubation, ethanol was added, the mash was centrifuged to recover thewet solids which were then dried and used in fish feeding trials. Bymonitoring performance of the conversion process, the yield andcomposition of the HQSPC, several parameters were observed thatsignificantly affected solids recovery. In the large scale trials, theparameters as shown in Table 1 were varied.

TABLE 1 Pre-Pilot scale trail variables and key performance parameters.Parameter Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6 Extruded yesyes no no no yes Sacc Time (h) 3 3.5 4 7 5 5 Incub pH 4.2 5.1 4.3 4.63.05 3.15 Incub Temp 29-30 29-30 29-30 26-27 29-30 29-30 (C.) Aeration0.25 0.25 0.25 0.5 0.5 0.5 (L/L/min) Incub Time 16 15 3.5 4.5 2 2.5(days) Solids 20 16 48 48 60 64 Recovery (%) Solids % 75.18 75.04 63.9361.5 61.61 56.86 Protein Trypsin 0 0 NA NA 16,750 6,538 InhibitorsSupernatant 2.5 5 2.1 3.89 2.39 3.4 % Solids Supernatant 78.16 80 71.6661.47 NA NA % Protein

From the HQSPC yields and protein levels, the following were noted: 1)an incubation pH of 3-3.5 and temperature of 30-32° C., along with highaeration maximized growth of A. pullulans and minimized pullulanproduction, 2) an incubation time of 4-5 days was optimal for proteincontent and solids recovery, 3) longer incubation times increasedprotein content, but substantially reduced solids recovery, 4) shorterincubation times maintained high solids recovery, but limited proteincontent, and 5) due to the lack of stachyose and raffinose in the endproduct, extrusion and/or reduced (omitted) enzymatic saccharificationmay be possible.

Preliminary bench-scale trials in the 5 L bioreactors were conducted tooptimize process conditions. A 10% solid loading rate of extruded whiteflake was used and saccharified for 24 h, followed by inoculation withA. pullulans and incubated at pH 5, 0.5 L/L/min aeration, 200 rpmagitation for 10 days. The extended incubation time was tested toestablish optimal harvest window to maximize both percent solidsrecovery and protein content in the solids. Samples (100 ml) wereremoved daily and, on alternate days, were subjected to the following:

-   -   Precipitating all solids with ethanol, centrifuging and drying        solids, measuring residual solids in the resulting supernatant.    -   Centrifuging the broth first to recover solids, drying the        solids, precipitating the pullulan from the resulting        supernatant and drying.

The ethanol precipitation first method recovered about 97% of the solids(soybean solids, cells and gum) using a lab centrifuge (10,000 g), withabout 3% solids remaining in the fluid phase. The centrifugation firstmethod recovered about 81.7% of solids (soybean solids and cells), withethanol precipitation of the supernatant recovering about 14.8% solids(exopolysaccharide), and about 3.5% solids remaining in the fluid.

Through these bench scale trials, levels of protein, pullulan, and totalsolids that could be recovered each day were measured. It was expectedthat as incubation proceeded, protein and pullulan levels wouldincrease, but that total solids recovered would decrease as somenutrients were catabolized into water and CO₂. Average protein levels ofthe solids from three replications are shown in FIG. 3. Protein levelsreached 70% by day 3-5, while total solids recovered begin dropping byday 5-6. Thus it appears that a 4-5 day incubation time may be optimal.

Performance Evaluation of HQSPC as Fish Meal Replacement in Perch Fish

Several difference among commercially available SPC were previouslyidentified, principally in protein and amino acid composition andanti-nutritional properties, which imparted variability in growthperformance and fish composition. Those experiments justified the needto develop higher quality SPC products that would support growthperformance equivalent to or better than diets containing fish meal. Afeeding trial was conducted utilizing yellow perch to provide assessmentof two HQSPC soy products (fermentation trials 5 and 6) in comparison toa commercial SPC and a Menhaden fish meal control.

Feed Preparations: Seven diets were formulated as follows:

-   -   Diet 1=fish meal control    -   Diet 2=commercial SPC    -   Diet 3=commercial SPC (supplemented with lysine+methionine)    -   Diet 4=HQSPC trial 5    -   Diet 5=HQSPC trial 5 (supplemented with lysine+methionine)    -   Diet 6=HQSPC trial 6    -   Diet 7=HQSPC trial 7 (supplemented with lysine+methionine)

Approximately 12 kg of each diet were prepared, including 2 kgcontaining 1 g/100 g chromic oxide for digestability determinations. Thetrial diets were formulated to contain equivalent SPC amounts with anappropriate protein:lipid target of 42:10. Soy Protein Concentrate (SPC,e.g., from Netzcon Ltd. Rehovot, Israel) with a minimum protein contentof 69% is made by aqueous alcohol extraction of defatted non-toastedwhite flakes. SPC is distinctly different from soybean meal, as itcontains traces of oligopolysaccharides and antigenic substancesglycinin and b-conglycinin.

Large particle ingredients were ground with a Fitzpatrick comminutator(Elhurst, Ill.) with 0.51 mm screen prior to dry blending. Dryingredients were blended for 20 min using a VI-10 mixer with anintensifier bar (Vanguard Pharmaceutical Machinery, Inc., Spring, Tex.).Dry blended feedstuffs were then transferred to a Hobart HL200 mixer(Troy, Ohio) where oils and water were added and blended for about 5min. Feeds were then screw pressed using a Hobart 4146 grinder with a3/16″ die and dried under cool, forced-air conditions. Following drying,feeds were milled into pellets using a food processor, sieved to achieveconsistent pellet size, and placed in frozen storage at −20° C. Chemicalanalyses of primary protein sources may be seen in Table 2.

TABLE 2 Composition of the primary protein sources (g/100 g, dry matterbasis (dmb)) incorporated into yellow perch experimental diets. MenhadenCommercial HQSPC HQSPC Protein Source Fish Meal SPC Trial 5 Trial 6Proximate Components Protein 66.77 78.18 61.61 56.86 Moisture* 7.62 9.735.14 7.89 Lipid 5.21 0.00 1.70 1.26 Crude Fiber 0.18 10.08 0.81 4.86 Ash25.33 7.10 8.82 5.21 Amino Acids Alanine 3.97 3.03 2.71 2.66 Arginine3.69 5.30 2.44 3.65 Aspartic Acid 5.47 8.08 6.72 6.45 Cystine 0.48 0.970.87 0.88 Glutamic Acid 7.73 12.51 8.70 8.85 Glycine 4.81 2.96 2.67 2.51Histidine 1.26 1.84 1.41 1.40 Hydroxylysine 0.27 0.04 0.81 0.10Hydroxyproline 1.19 0.08 0.10 0.07 Isoleucine 2.73 3.30 2.89 2.92Lanthionine 0.00 0.02 0.00 0.00 Leucine 4.47 5.61 4.64 4.87 Lysine 4.584.56 3.47 3.41 Methionine 1.72 1.00 0.83 0.90 Ornithine 0.14 0.04 0.140.04 Phenylalanine 2.51 3.62 2.89 3.08 Proline 3.31 3.65 3.17 2.92Serine 1.85 3.07 2.28 2.73 Taurine 0.42 0.08 0.09 0.10 Threonine 2.322.80 2.36 2.31 Tryptophan 0.58 1.00 0.79 0.82 Tyrosine 2.01 2.57 1.982.25 Valine 3.10 2.51 3.13 3.10 Oligosaccharides Raffinose — 0.00 0.000.00 Stachyose — 0.24 0.00 0.00 Phytic Acid — 0.23 0.39 0.18 *Allingredients are expressed on a dry matter basis with the exception ofmoisture (as is). Analyses were for crude protein (AOAC 2006, method990.03), crude fat (AOAC 2006, method 9903), crude fiber (AOAC 2006,method 978.10), moisture (AOAC 2006, method 934.01) Chromic oxide (AOAC2006, method 990.08), ash (AOAC 2006, method 942.05), and amino acids(AOAC 2006, method 982.30 E(a,b,c)).

Pellet Properties

Samples of each diet were analyzed in triplicate for moisture (%), wateractivity (a_(w)), unit density (kg/m3), pellet durability index (%),water stability (min), and color (L, a, b); compressive strength (g),and diameter (mm) were determined with n=10 replications. Moisture (%)was obtained using standard method 2.2.2.5 (NFTA, 2001). Water activity(a_(w)) of 2 g pellet samples was measured with a Lab Touch a_(w)analyzer (Nocasina, Lachen S Z, Switzerland). Three color variables wereanalyzed with a spectrophotocolorimeter (Lab Scan XE, HunterLab, Reston,Va.) as Hunter L (brightness/darkness), Hunter a (redness greenness) andHunter b (yellowness/blueness). Unit density (UD) was estimated byweighing 100 ml of pellets and dividing the mass (kg) by 0.0001 m³.Pellet durability index (PDI) was determined according to standardmethod 5269.4 (ASAE 2003). The PDI was calculated as: PDI(%)=(M_(a)/M_(b))×100, where M_(a) was the mass (g) after tumbling andM_(b) was the mass (g) before tumbling. Pellet stability (min) wasdetermined by the static (W_(static)) method (Ferouz et al., Cereal Chem(2011) 88:179-188) to mimic pellet leaching in tanks until they wereconsumed. Stability was calculated as loss of weight from leaching/dryweight of initial sample. Pellet diameter was measured using aconventional caliper. Pellets were tested for compressive strength usinga TA.XT Plus Texture Analyzer (Scarsdale, N.Y.).

Feeding Trial

Yellow perch (2.95 g±0.05 SE) were randomly stocked at 21 fish/tank into28 circular tanks (110 liters) connected in parallel to a closed-looprecirculating aquaculture system (RAS). The RAS water flow and qualitywas maintained with a centrifugation pump consisting of dual solids suptanks, bioreactor, bead filter, UV filter, and heat pump. System waterwas municipal that is dechlorinated and stored in a 15,200 L tank. Fourreplications of each treatment were applied randomly in tanks. Waterflow was maintained at −1.5 L/min/tank. Temperature was maintained at22° C.±1°. Temperature and dissolved oxygen were measured with a YSI ProPlus (Yellow Springs Instrument Company, Yellow Springs, Ohio).Ammonia-nitrogen, nitrite-nitrogen, nitrate-nitrogen, alkalinity (asCaCO₃), and free chlorine were tested using a Hach DR 3900Spectrophotometer (Hach Company, Loveland, Colo.).

Fish were fed to satiation by hand twice daily, and feeding rates weremodified according to tank weights, observed growth rates, and feedconsumption assessments. Consumption (%) was estimated from a knownnumber of pellets fed and by counting uneaten pellets 30 min afterfeeding. Collections of uneaten feed with subsequent dry weights werealso used to estimate consumption. Weekly tank consumption estimateswere multiplied by weekly rations to obtain weekly consumption (g).Palatability of treatments was determined by the amount of feed consumedor rejected. Tank mass (+0.01 g) was measured every other week to adjustfeed rates and calculate performance indices. Individual lengths (mm)and weights (+0.01 g) were also measured every other week on fourrandomly sampled fish from each treatment.

Feed conversion ratio (FCR) was calculated as:

${F\; C\; R} = \frac{{mass}\mspace{14mu}{of}\mspace{14mu}{feed}\mspace{14mu}{consumed}\mspace{14mu}\left( {{dry},g} \right)}{{growth}\mspace{14mu}\left( {{wet},g} \right)}$

Protein conversion ratio was calculated as:

${PER} = \frac{{growth}\mspace{14mu}\left( {{wet},\; g} \right)}{{mass}\mspace{14mu}{of}\mspace{14mu}{protein}\mspace{14mu}{consumed}\mspace{14mu}\left( {{dry},g} \right)}$

Fulton-type condition factor (K) was calculated as:

$K = {\frac{{weight}\mspace{14mu}(g)}{\left\lbrack {{length}\mspace{14mu}({mm})} \right\rbrack^{3}} \times 10,000}$

Specific growth rate (SGR) was calculated as:

${S\; G\; R} = \frac{\left\lbrack {{{In}\left( {{final}\mspace{14mu}{{wt}(g)}} \right)} - {{In}\left( {{start}\mspace{14mu}{{wt}(g)}} \right)}} \right\rbrack \times 100}{n({days})}$

Statistical analyses of diets and feeding trial responses were carriedout with analysis of variance (ANOVA, a priori α=0.05). Significant Ftests were followed by a post hoc Tukey's test to separate treatmentmeans.

Pellet and Feed Results

Feed formulations were based on HQSPC nutrient analyses from Trail 3(Table 1), while all soy protein concentrates were included equally at45% (100% fish meal replacement) in trial diets. Analyses of Trials 5and 6 were completed after scheduled feeding trial start date (Table 2),generally resulting in similar but not isonitrogenous diets. Diets wereformulated to contain 42% protein and 10% lipid, with energy-to-protein(E:P) ratios of 7.91 to 7.94 (kcal/g). Recent analyses showed that crudeprotein (dmb) was 44.9% (Diet 1), 43.2% (Diets 2 and 3), 36.8% (Diets 4and 5), and 37.5% (Diets 6 and 7). Crude lipid was approximately 10% forall diets. Amino acid analysis of feeds did not reveal any potentialdeficiencies among un-supplemented diets compared to yellow perchrequirements (Table 3).

TABLE 3 Experimental design, dietary formulations, and compositions forthe perch feeding trials. Diet # Ingredients (g/100 g, dmb) 1 2 3 4 5 67 Menhaden Fish Meal^(a) 50.0 0.0 0.0 0.0 0.0 0.0 0.0 Commercial SPC 0.045.0 45.0 0.0 0.0 0.0 0.0 HQSPC Trial 5 0.0 0.0 0.0 45.0 45.0 0.0 0.0HQSPC Trial 6 0.0 0.0 0.0 0.0 0.0 45.0 45.0 Yellow Corn Gluten^(b) 5.05.0 5.0 5.0 5.0 5.0 5.0 Wheat Flour^(c) 18.0 21.0 21.0 21.0 21.0 21.021.0 Wheat Gluten^(b) 6.0 5.0 5.0 5.0 5.0 5.0 5.0 CMC^(d) 5.0 5.0 5.05.0 5.0 5.0 5.0 Celufil^(d) 3.7 3.6 3.6 3.5 3.0 3.5 3.0 Menhaden Oil^(e)4.59 8.19 8.19 8.37 8.37 8.37 8.37 Flax Oil^(f) 0.51 0.91 0.91 0.93 0.930.93 0.93 Vitamin Premix^(g) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 MineralPremix^(h) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 Vitamin C^(i) 0.05 0.05 0.05 0.050.05 0.05 0.05 Choline^(j) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Phytase^(k) 0.0370.037 0.037 0.037 0.037 0.037 0.037 Brewer's Yeast^(l) 2.0 2.0 2.0 2.02.0 2.0 2.0 L-Lysine^(j) 0.0 0.0 0.3 0.0 0.3 0.0 0.3 L-Betaine^(j) 0.50.5 0.5 0.5 0.5 0.5 0.5 L-Methionine^(j) 0.0 0.0 0.2 0.0 0.2 0.0 0.2Sodium Chloride^(m) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Potassium Chloride^(m)0.8 0.8 0.8 0.8 0.8 0.8 0.8 Magnesium Oxide^(m) 0.05 0.05 0.05 0.05 0.050.05 0.05 Calcium Phosphate^(m) 2.0 1.0 1.0 1.0 1.0 1.0 1.0 EstimatedProximate Composition Protein (%) 42.1 42.2 42.7 42.0 42.5 42.0 42.5Lipid (%) 10.06 10.01 10.01 10.03 10.03 10.03 10.03 Ash (%) 4.46 3.463.46 3.46 3.46 3.46 3.46 Total Energy (kcal) 334.3 334.3 337.1 333.5336.3 333.5 336.3 E:P (kcal/g) 7.94 7.93 7.94 7.91 7.94 7.94 7.91^(a)Special Select, Omega Protein, Houston, TX; ^(b)Consumers SupplyDistribution, Sioux City, IA; ^(c)Bob's Red Mill Natural Foods,Milwaukie, OR; ^(d)USB Corporation, Cleveland, OH; ^(e)Virginia PrimeGold, Omega Protein, Houston TX; ^(f)Thomas Laboratories, Tolleson, AZ;^(g)ARS 702 Premix, Nelson and Sons, Murray, UT; ^(h)SS #3 Trace Mix,Nelson and Sons, Murray, UT; ^(i)U.S. Nutrition, Bohemia, NY; ^(j)PureBulk, Roseburg, OR; ^(k)DSM Nutritional Products, Parsippany, NJ;^(l)Diamond V Mills, Cedar Rapids, IA; ^(m)Fisher Scientific, Pittsburg,PA.

Pellet feeds exhibited significant differences in measurements amongtreatments except for diameters (Table 4).

TABLE 4 Physical properties of the feed extrudates. Diet # Prop- erties1 2 3 4 5 6 7 MC 8.5 ± 11.0 ± 11.0 ± 14.1 ± 9.9 ± 10.1 ± 12.2 ± (% db)0.2 ^(a) 0.3 ^(b) 0.1 ^(b) 0.1 ^(c) 0.1 ^(d) 0.1 ^(d) 0.1 ^(e) a_(w) (−)0.58 ± 0.67 ± 0.69 ± 0.68 ± 0.68 ± 0.68 ± 0.74 ± 0.02 ^(a) 0.00 ^(b)0.00 ^(c) 0.00 ^(bc) 0.00 ^(bc) 0.00 ^(bc) 0.01 ^(d) BD 634.8 ± 648.6 ±659.4 ± 675.4 ± 688.9 ± 669.3 ± 695.9 ± (kg/m³) 3.0 ^(a) 3.2 ^(b) 2.4^(bc) 0.5 ^(d) 0.6 ^(e) 2.8 ^(cd) 2.2 ^(e) CS (g) 24.4 ± 56.1 ± 42.9 ±44.9 ± 54.8 ± 67.0 ± 60.1 ± 0.7 ^(a) 1.8 ^(b) 2.3 ^(c) 3.8 ^(bc) 2.3^(b) 2.2 ^(b) 3.9 ^(b) PDI (%) 98.1 ± 98.1 ± 98.1 ± 98.3 ± 99.3 ± 99.5 ±99.5 ± 0.4 ^(a) 0.8 ^(a) 0.7 ^(a) 0.3 ^(a) 0.3 ^(b) 0.2 ^(b) 0.3 ^(b)WSI_(still) 10.2 ± 11.9 ± 9.1 ± 8.9 ± 9.1 ± 8.2 ± 6.7 ± (%) 0.0 ^(ab)0.0 ^(a) 0.0 ^(ab) 0.0 ^(ab) 0.0 ^(ab) 0.0 ^(ab) 0.0 ^(b) L (−) 47.9 ±58.5 ± 68.5 ± 59.9 ± 53.7 ± 60.6 ± 63.8 ± 0.2 ^(a) 0.1 ^(b) 0.5 ^(c) 0.3^(bd) 0.2 ^(e) 0.3 ^(d) 0.4 ^(f) a (−) 5.2 ± 2.8 ± 3.2 ± 4.3 ± 4.4 ± 2.9± 2.9 ± 0.0 ^(a) 0.0 ^(b) 0.1 ^(c) 0.0 ^(d) 0.0 ^(d) 0.0 ^(b) 0.0 ^(b) b(−) 22.8 ± 18.1 ± 20.2 ± 22.8 ± 21.4 ± 20.3 ± 20.7 ± 0.1 ^(a) 0.1 ^(b)0.1 ^(c) 0.1 ^(a) 0.1 ^(d) 0.0 ^(ce) 0.1 ^(e) Dia. 2.2 ± 2.1 ± 2.1 ± 2.0± 2.0 ± 2.0 ± 2.0 ± (mm) 0.0 ^(a) 0.1 ^(a) 0.0 ^(a) 0.0 ^(a) 0.0 ^(a)0.0 ^(a) 0.0 ^(a) Values given are means (±SE) associated with treatmentmeans. Values not significantly different (P > 0.05) have same letterwithin a given row. MC (% db) = moisture content; a_(w) (−) = wateractivity; BD (kg/m³) = unit density; CS (g) = compressive strength; PDI(%) = pellet durability index; WSI_(still) (%) = water solubility indexin still water; L (−) = Hunter brightness; a (−) = Hunter yellowness; b(−) = Hunter redness; Dia. (mm) = diameter.

Moisture content (MC) ranged from about 8.49% (Diet 1) to about 14.07%(Diet 4). MC contributes an effect on other characteristics, such asPDI, compressive strength, and color. No apparent correlation between MCand other variables were identified.

Water activity, a measure of unbound water in pellets, was high (about0.58 to about 0.74), with Diet 1 (fish meal) significantly lower (about0.58) and Diet 7 significantly higher (about 0.74) than all othertreatments. Values over 0.6 indicate low storage stability and may allowmicrobial growth to proliferate. Feeds were stored in a freezer at −20°C.

Unit density (BD), a measure of feed weight per unit volume, ranged fromabout 634.87 to about 695.9. Diet 1 (fish meal) had a lower DB, andwhile not being limited by theory, this is most likely due to lowerinclusion of fish oil and fish meal. Soy protein concentrate diets hadhigher BDs because they contained more oil to amend lipid requirements.

Compressive strength (CS) was calculated as peak fracture force of thestress-strain curve from a perpendicular axial direction. CS variedsignificantly from about 24.36 to about 67.03. Diet 1 (fish meal)exhibited the lowest compressive strength (about 24.36). While not beinglimited by theory, this is most likely attributable to the heterogeneityof the pellets, which results from the pelleting process (e.g., screwpressed rather than extruded). Extrusion cooks the feed with acombination of moisture, pressure, temperature, and mechanical shear.This process gelatinizes starches, which can increase CS substantially.

Pellet Durability Index (PDI) was very high in all diets (about 98.05%to about 99.48%), with no significant difference between treatments.These high PDI values may be the result of high MC, as well ascarboxymethylcellulose (CMC) binder addition.

Water Solubility Index (WSI) was low in all diets (about 9.65 to about14.43), with Diet 4 exhibiting the highest value, which wassignificantly different than the lowest, Diet 7, at about 9.65. Due tothe nature of screw-pressed compared to extruded feeds, WSI was expectedto be low. Extruded feeds are more water stable due to gelatinization ofstarches, reducing water penetration.

Hunter color parameters (L, a, b) revealed similarities with growthperformance. Hunter a (redness) was highest in diet 1 (5.15) and lowestin Diet 2 (2.80), Hunter b (yellowness) was highest in Diet 4 (22.81)and Diet 1 (22.76) and lowest in Diet 2 (18.07). Hunter L (brightness)was highest in Diet 3 (68.48) and lowest in Diet 1 (47.91). Lightercolor feeds have been found to contain higher concentrations and greateravailability of lysine compared to darker feeds.

Fish Performance

Diet 6 provided the most comparable results to Diet 1 (fishmeal) ingrowth performance. Growth performance was significantly differentbetween treatments in all categories (Table 5).

TABLE 5 Performance Aspects. Diet WG SGR S TC FCR PER K 1 102.1 ± 1.66 ±100.0 ± 450.2 ± 1.27 ± 1.76 ± 1.06 ± 8.0 ^(a) 0.1 ^(a) 0.0 ^(a) 5.8 ^(a)0.05 ^(a) 0.06 ^(a) 0.01 ^(ab) 2 12.6 ± 0.55 ± 82.3 ± 140.9 ± 2.59 ±0.98 ± 1.01 ± 6.4 ^(b) 0.12 ^(b) 0.6 ^(b) 4.7 ^(b) 0.49 ^(b) 0.17 ^(b)0.02 ^(a) 3 23.7 ± 0.59 ± 96.4 ± 181.6 ± 2.0 ± 1.17 ± 1.02 ± 1.3 ^(bc)0.04 ^(bc) 0.3 ^(a) 2.5 ^(bc) 0.09 ^(ab) 0.06 ^(b) 0.03 ^(ab) 4 46.6 ±1.06 ± 94.1 ± 283.4 ± 1.6 ± 1.7 ± 1.11 ± 8.3 ^(cd) 0.12 ^(c) 0.5 ^(abc)8.9 ^(bc) 0.04 ^(a) 0.04 ^(a) 0.04 ^(ab) 5 49.1 ± 1.10 ± 92.9 ± 306.3 ±1.73 ± 1.59 ± 1.14 ± 11.0 ^(cd) 0.14 ^(d) 0.3 ^(abc) 11.2 ^(ac) 0.10^(ab) 0.10 ^(a) 0.03 ^(b) 6 84.4 ± 1.48 ± 98.8 ± 451.5 ± 1.43 ± 1.87 ±1.07 ± 2.4 ^(ae) 0.05 ^(ad) 0.3 ^(ac) 13.4 ^(a) 0.04 ^(a) 0.05 ^(a) 0.03^(ab) 7 61.8 ± 1.28 ± 94.1 ± 338.1 ± 1.58 ± 1.69 ± 1.06 ± 4.8 ^(de) 0.12^(ad) 0.6 ^(abc) 10.8 ^(ac) 0.03 ^(a) 0.03 ^(a) 0.02 ^(ab) Mean weightgain (WG, %), specific growth rate (SGR), survival (S, %), totalconsumption (TC, g), food conversion ratio (FCR), protein efficiencyratio (PER), and Fulton-type condition factor (K) values for perch fedexperimental diets. Values given are means (±SE) associated with thetreatment means. Values not significantly different (P > 0.005) have thesame letter within a given column.

Supplemented diets (Diets 3 and 5) were slightly better than theirun-supplemented counterparts, except for Diets 6 and 7. Diet 6outperformed Diet 7 in all aspects. While not being bound by theory,this could be the result of timing issues associated with thebioavailability of crystalline amino acid supplements.

Weight Gain (WG) was highest for Diet 1 (fishmeal at 102.1%), followedby Diets 6 and 7 (84.4% and 61.8%, respectively). Diets 2 and 3(commercial SPC) exhibited the lowest WG (12.62% and 23.69%,respectively). Specific Growth Rate (SGR) paralleled WG results. SGR washighest in Diet 1 (1.66) and lowest in Diet 2 (0.55).

Protein Efficiency Ratio (PER) was highest in Diet 6 (1.87), followed byDiet 1 (1.76) and Diet 4 (1.70). Diets 2 and 3 had the lowest PERs of0.98 and 1.17, respectively. PER was better in un-supplemented Diets 4and 6 than in supplemented diets 5 and 7.

Feed conversion ratio (FCR) followed a similar pattern, with Diets 1 andDiet 6 being lowest (1.39 and 1.59, respectively). These FCR valuesindicate very high nutritional feed quality. Diets 2 and 3 had thehighest FCR (2.91 and 2.24, respectively).

Total consumption (dmb) was highest with Diet 6 (451.5 g) and Diet 1(450.20 g) and lowest with Diet 2 (140.91) and Diet 3 (181.60). Alldiets were fed to satiation. Diets consumption was deemed indicative ofpalatability, which is also the reason for significantly lower survivalfor fish on Diet 2 (82.29%). Survival was 100% for Diet 1 and 98.81% forDiet 6.

Fulton's Condition Factor (K) was highest for Diet 5 (1.14) and lowestfor Diet 2 (1.01). All HQSPC diets met or exceeded the fishmeal controlfor this specific performance parameter. Commercial SPC diets were lowerthan all other diets.

Other Assays

End of trial analyses may include final growth, FCR, PER, consumption,and examination for nutrition deficiencies via necropsy. Plasma assaysmay be completed for lysine and methionine using standard methods.Individual fish may be euthanized by cervical dislocation in order toquantify muscle ratio, hepatosomatic index, viscerosomatic index, filletcomposition, and hind gut histology (enteritis inflammation scores).Protein and energy availability of trial diets may be estimated usingchromic oxide (CrO₃) marker within the feed and fecal material. Fecalmaterial may be collected via necropsy from the lower intestinal tract.

The apparent digestibility coefficients (ADC) for the nutrients in thetest diets may be calculated using the following formula:

${ADC}_{{test}\mspace{14mu}{ingredient}} = {{ADC}_{{test}\mspace{14mu}{diet}} + \left\lbrack {\left( {{ADC}_{{test}\mspace{14mu}{diet}} - {ADC}_{{ref}\mspace{14mu}{diet}}} \right) \times \left( \frac{0.7 \times D_{ref}}{0.3 \times D_{ingr}} \right)} \right\rbrack}$

where Dref=% with nutrient (kJ/g gross energy) of reference diet mash(as is) and Dingr=% nutrient (kJ/g gross energy) of test ingredient (asis).

SUMMARY

Screw-pressed compound feeds for yellow perch were exemplified using twoHQSPC diets, a fish control meal, and a commercial soy proteinconcentrate (SPC). The HQSPC-based diets had comparable performance tofish meal and outperformed the commercial SPC. Growth and conversionperformance exceeded expectations, given crude protein content in theHQSPC diets was approximately 7% less than the fish meal control and 6%less than the commercial SPC. Thus, the HQSPC diets may serve as acomplete replacement for fish meal.

Performance evaluation of HQSPC as Fish Meal Replacement in RainbowTrout

In addition to the yellow perch results above, a 90-day feeding trialwas conducted using a domesticated rainbow trout strain (Shasta). Table6 summarizes the protein targets by fish species and size. These dietaryprotein targets or levels (%) may be used in the formulation ofexperimental diets for these commercially important finfish.

TABLE 6 Protein targets for various finfish by weight range. Weightranges (g) Species <20 20-200 200-600 600-1,500 >1,500 Atlantic salmon48 44 40 38 34 Pacific salmon 55 45 40 38 38 Rainbow trout 48 40 38 3836 Channel catfish 44 36 32 32 28 Nile tilapia 40 34 30 28 26

Trout were fed a control fish meal diet or a diet replacing 70% of fishmeal (Diet 2—35% inclusion) with an HQSPC product (Trial 6) (see Table7).

TABLE 7 Test Diet Formulations Used in a Rainbow Trout Feeding TrialIngredient (g) Diet 1 Diet 2 Omega prime special select 40 15 HQSPC 0 35Yellow corn gluten 20 16 Wheat flour 15 11 Carboxymethylcellulose 6.7 3Vitamin premix 1.5 1.5 Trace mineral premix 1.5 1.5 Stay-C 0.5 0.5DVAqua 0.1 0.1 Methionine 0 0.2 Sodium chloride 0.5 0.5 Potassiumchloride 0.5 0.5 Magnesium oxide 0 0 Calcium phosphate 0 0.5 Menhadenoil 13.7 14.7 Proximates Protein 42.87 42.89 Fat 16.37 16.38 Fiber 0.612.14 Ash 12.31 8.15 Kcal 398.54 398.68 E:P 9.30 9.29

The 35% inclusion level was used to exceed the 30% inclusion recommendedfor other soy protein concentrates, such as Selecta SPC60. This trialusing isocaloric, isonitrogenous diets demonstrated equivalentperformance between the control and test diet provided.

Table 8 summarizes the observed rainbow trout performancecharacteristics during the 90-day feeding trial. Diet 2, containingHQSPC, showed no decline in weight gain or feed conversion/efficiencyand there were no mortalities (100% survival). These observationsreplicate the results observed in the yellow perch trials.

TABLE 8 Performance trails for rainbow trout using HQSPC. FM:HQSPC (%)Diet 1 (40:0) Diet 2 (15:35) Start weight (g) 795 + 67 882 ± 44 Endweight (g) 3,400 ± 216 3,706 ± 172 Gain (g) 2,605 ± 149 ab 2,824 ± 133 aGain (%) 327.7 320.2 Food fed (g) 3,608 ± 189 3,880 ± 68 FCR 1.39 ± 0.06a 1.38 ± 0.07 a Mortality (%) 0.0 + 0.0 0.0 + 0.0 Values given are means(± SE) associated with the treatment means.

SUMMARY

Screw-pressed compound feeds for rainbow trout were exemplified using anHQSPC diet and a fish control meal. In this trial the HQSPC-based dietshad comparable performance to fish meal. Again, these data show that theHQSPC diets exceeded the expected results using SPC (e.g., HQSPC may beused at concentration greater than a 30% inclusion level), and may serveas an efficacious replacement for fish meal.

Example 2. Production of HP-DDGS Using Microbial Conversion

The effects of extrusion on improving saccharification of DDGS using asingle screw extruder (BRABENDER PLASTI-CORDER EXTRUDER Model PL2000,Hackensack, N.J.) with barrel length to screw diameter of 1:20 and acompression ratio of 3:1 was investigated (FIGS. 4 and 5). It wasdetermined that 25% DDGS moisture content, temperature of 100° C. to160° C., and screw speed of 200 rpm resulted in a 36% sugar recoveryfrom corn fiber (FIG. 5). The performance of various NOVOZYMElignocellulose deconstructing enzymes was separately evaluated and itwas found that 6% CELLIC CTEK2 (per gm glucan) and 0.3% CELLIC HTEK2(per gm total solids) resulted in sugar recoveries up to 70%. Thesepretreatment and saccharification conditions may be used to generateHP-DDGS. Next options such as co-culturing with cellulase-producers toreduce the need for added enzymes may be carried out, as well as usingfed-batch bioreactors to reduce processing costs.

Evaluating growth and gum production of the microbe on the carbohydratesfound in soybean meal was carried out and it was found that proteincontent can be increased from 42% to at least 60% by using the approachas disclosed herein. This showed that the microbe (e.g., A. pullulans)can efficiently convert a broad range of difficult to metabolizeoligosaccharides into cell mass (i.e., protein) and a microbial gum.This effort with soybean meal was initiated based on prior studies thatevaluated production of a range of microbial gums (exopolysaccharides)from corn processing byproducts such as whole stillage, thin stillage,and condensed corn solubles. Through this work, a variety of microbialstrains have been accumulated that efficiently grow on various cornprocessing byproducts and produce high levels of cell mass and gums fromthe available sugars. Key operational parameters and lower-cost gumrecovery methods have been identified and developed. Based on this bodyof work, and the knowledge that the strains of microbes produce a broadrange of hydrolytic enzymes, robust processes have been identified thatallow for effective conversion DDGS into HP-DDGS (e.g., A. pullulansstrain NRRL No. 50793).

For pretreatment, the conventional, dryfrac, and/or low oil DDGS areextruded in a single screw extruder (BRABENDER PLASTI-CORDER EXTRUDERModel PL2000, Hackensack, N.J.) with a barrel length to screw diameterof 1:20 and a compression ratio of 3:1. DDGS samples (adjusted to 25%moisture), the temperature of feed, barrel, and outlet sections ofextruder is held at 100° C. to 160° C., and screw speed is set at 200rpm, providing a shearing effect against the ridged channels on bothsides of the barrel. These selected levels of temperature, screw speedand moisture were based on optimized conditions defined previously thatresulted in 36% sugar release from corn fiber due to disruption of theDDGS matrix.

Extruded conventional and dryfrac DDGS is mixed with water to achieve asolid loading rate of at least 5% in a 5 L NEW BRUNSWICK BIOFLO 3BIOREACTOR (3-4 L working volume) at a pH of 5.8. After autoclaving andcooling, the slurry is saccharified using a cocktail of NOVOZYME enzymesfor which preliminary data has previously been collected. Dosages to beused in the initial trials includes 6% CELLIC CTEK2 (per gm glucan) and0.3% CELLIC HTEK2 (per gm total solids). Saccharification is conductedfor 24 h at 50° C. and 150 rpm to solubilize the fibers andoligosaccharides into simple sugars. The temperature is then reduced to35° C., the pH is adjusted to 4.0 (to optimize cell growth), and theslurry is inoculated with 2% (v/v) of a 24 h culture of the microbe. Theslurry is aerated at 0.5 L/L/min and incubation is continued until sugarutilization ceases (96-120 h anticipated). The following parameters arethen evaluated:

-   -   1) replacing the cellulase enzymes with cellulase producing        microbes that would be co-cultured with the microbe;    -   2) maximizing initial solid loading rate; and    -   3) adding more extruded substrate during either saccharification        and/or the microbial conversion phases (i.e., fed-batch        operation) to minimize net enzyme dosage, maximize protein and        gum concentrations, and minimize product recovery costs.

During incubation, samples are removed at 6-12 h intervals. Samples forHPLC analysis are boiled (to inactivate enzymes), centrifuged, filteredthrough 0.22-μm filters, placed into autosampler vials, and frozen untilanalysis. These samples are assayed for carbohydrates and organicsolvents using a WATERS HPLC system. Samples are subjected to microbialcounts to assess microbial populations. Samples are also assayed forlevels of cellulose and hemicellulose using National Renewable EnergyLaboratory procedures.

The converted slurry is then subjected to ethanol precipitation andcentrifugation to separate the protein, microbial gum and microbialbiomass (HP-DDGS) from the remaining culture fluid. While not beingbound by theory, the presence of a precipitating gum improves theefficiency of centrifugation in recovering suspended solids. Thecomposition of the HP-DDGS is then determined and used in fish feedingtrials. Ethanol is recovered from the liquid stream via distillation,and the residual liquid is chemically analyzed to assess potential uses(e.g., incorporation into the HP-DDGS or biogas production).

The HP-DDGS so produced contains a microbial gum to serve as a bindingagent and potentially as an immunostimulant. Other hydrolytic enzymesexcreted by the microbe should release peptides, amino acids, and anyremaining lipids, thereby increasing feed intake, growth performance,and nutrient utilization in fish feeding trials.

Evaluation of the Performance of HP-DDGS as a Fish Meal Replacement inPerch Feeds

Conventional and dryfrac HP-DDGS from the above are analyzed fornutritional competencies in view of requirements of targeted species,especially focusing on yellow perch. Samples will be subjected tochemical analyses: proximate analysis, Van Soest fibers, insolublecarbohydrates, amino acids, fatty acids, and minerals. This ensures thatnutritional benchmarks have been satisfied. Any anti-nutritionalproperties (e.g., phytic acid content) of HP-DDGS are compared withcurrent DDGS and provide a basis for further process modification.

Complete practical diets are formulated using conventional and dryfracHP-DDGS in accordance with known nutrient requirements for yellow perch(e.g., 32% protein, 8% lipid). Basal mineral and vitamin premixes forplant-based diets are used to ensure that micro-nutrient requirementsare met. Any supplements (as deemed necessary by analysis) are evaluatedby comparing to an identical formulation without supplementation; thus,the feeding trial is done in a factorial design to account forsupplementation effects. All feeding trials include a fish meal-basedcontrol diet and diets containing graded levels of HP-DDGS. Pellets forfeeding trials are produced using the lab-scale single screw extruder(BRABENDER PLASTI-CORDER EXTRUDER Model PL2000).

Replication of four experimental units per treatment are used in allfeeding trials (60 to 120 days each). Trials are done in 110-L circulartanks (20 fish/tank) connected in parallel to a closed-looprecirculation system consisting of a solids sump, bioreactor, andfilters (100 μm bag, carbon and ultra-violet) driven by a centrifugalpump. Heat pumps are used as required to maintain optimal temperaturesfor species-specific growth. Water quality (e.g., dissolved oxygen, pH,temperature, ammonia and nitrite) is monitored in all systems.

Experimental diets are delivered according to fish size, split intotwo-to-five daily feedings. Growth performance is determined by totalmass measurements taken at one-to-four weeks (depending upon fish sizeand trial duration); rations are adjusted in accordance with gains toallow satiation feeding and to reduce waste streams. Consumption isassessed biweekly from collections of uneaten feed from individualtanks. Uneaten feed is dried to a constant temperature, cooled, andweighed to estimate conversion efficiency. Protein, energy, andphosphorous digestibility are determined from fecal material manuallystripped during the midpoint of each experiment or via necropsy from thelower intestinal tract at the conclusion of a feeding trial. Survival,weight gain, growth rate, health indices, feed conversion, protein andenergy digestibility, protein efficiency, and phosphorous utilizationare compared among treatment groups. Proximate analysis of necropsiedfish is done to compare composition of fillets among dietary treatments.Analysis of amino and fatty acids is done as needed for filletconstituents, according to the feeding trial objective.

Statistical analyses of diets and feeding trial responses are completedwith an a priori α=0.05. Analysis of performance parameters amongtreatments is done with appropriate analysis of variance or covariance(Proc Mixed) and post hoc multiple comparisons, as needed. Analysis offish performance and tissue responses are assessed by nonlinear models.

Determining Preliminary Mass Balance, Energy Requirements, and Costs.

Inputs and outputs of the conventional and dryfrac HP-DDGS conversionprocess are monitored and used to establish a process mass balance.Similarly, energy requirements for the process are measured and/orestimated to calculate total energy use. Together, these inputs are usedto assess preliminary costs, which are compared to conventional anddryfrac HP-DDGS value.

All of the references cited herein are incorporated by reference intheir entireties.

From the above discussion, one skilled in the art can ascertain theessential characteristics of the invention, and without departing fromthe spirit and scope thereof, can make various changes and modificationsof the embodiments to adapt to various uses and conditions. Thus,various modifications of the embodiments, in addition to those shown anddescribed herein, will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

We claim:
 1. A composition comprising a soy protein concentrate fromsoybean containing feedstock, wherein the composition contains at leastabout 75% protein content by weight, at least one microbe, pullulan andcontains reduced oligosaccharides compared to the soybean containingfeedstock from which the composition is derived.
 2. The composition ofclaim 1, wherein the oligosaccharides are present in an amount ofbetween about 0.00 g/100 g to about 0.24/100 g on a dry matter basis. 3.The composition of claim 1 produced by a process comprising: a)pretreating soybeans by solvent washing, extruding, adding one or morecellulose deconstruction enzymes or a combination thereof to form amash; b) inoculating the mash with at least one microbe, and incubatingthe mash for a temperature and time sufficient to reduce theconcentration of anti-nutritional components of the soybeans; c)separating solids from liquids in the inoculated mash; and d) dryingsaid separate solids.
 4. The composition of claim 1, wherein the atleast one microbe is an Aureobasidium pullulan strain.
 5. Thecomposition of claim 4, wherein the A. pullulans strain is selected fromthe group consisting of NRRL No. 50792, NRRL No. 50793, NRRL No. 50794,NRRL No.
 50795. 6. The composition of claim 3, wherein the compositionexhibits a light color indicative of low caramelization and fewerMillard reactions of the free amino groups and available sugars comparedto the same composition having a darker color.
 7. The composition ofclaim 3, wherein the extruded soy feedstock is from soybeans in the formof soy flakes or soybean meal, wherein when said composition is fed tofish as a component of a feed, weight gain, specific growth rate (SGR),feed intake, feed conversion ratio (FCR) or combination thereof observedfor said fish are improved compared to fish fed non-extruded feedstock.8. The composition of claim 1, wherein the composition is contained in afeed or foodstuff, wherein the combination comprises phytase and whereinthe feed or foodstuff comprises reduced phytic acid compared to thesoybeans from which it is derived.
 9. A feed or foodstuff comprising thecomposition of claim
 1. 10. The feed or foodstuff of claim 9, whereinsaid feed provides about 35% to about 100% of the protein source in saidfeed.
 11. The feed or foodstuff of claim 10, wherein said composition isa complete replacement for animal-protein in a feed or foodstuff. 12.The feed or foodstuff of claim 9, wherein said feed or foodstuff isformulated for fish, crustaceans, cattle, poultry, swine, goats, sheep,cats, dogs, horses, or a combination thereof.
 13. The feed or foodstuffof claim 9, wherein said foodstuff is formulated for humans.
 14. Thecomposition of claim 1, wherein soybeans in the soybean containingfeedstock consists of GMO, non-GMO soybeans or a combination thereof.15. A method of producing a soy protein concentrate comprising: a)pretreating the soybeans by solvent washing, extruding, adding one ormore cellulose deconstruction enzymes or a combination thereof to form amash; b) inoculating the mash with at least one microbe, and incubatingthe mash for a time and temperature sufficient to reduce theconcentration of heat-labile and non-heat labile anti-nutritionalcomponents of the soybeans; c) separating solids from liquids in theinoculated mash; and d) drying said separate solids, wherein theresulting product of steps (a)-(d) contains at least about 75% proteincontent by weight, at least one microbe, pullulan and contains reducedoligosaccharides compared to the soybeans from which said concentrate isderived.
 16. The method of claim 15, wherein the solids are separated byat least one decanting centrifugation step, and wherein theanti-nutritional components are selected from the group consisting ofoligosaccharides, phytic acid, trypsin inhibitors and combinationsthereof.
 17. The method of claim 16, further comprising solvent washingand centrifuging the separated solids.
 18. The method of claim 15,wherein the one or more enzymes is selected from the group consisting ofphytase, endo-xylanase, and β-xylosidase, glycoside hydrolase,β-glucosidases, hemicellulase activities, and combinations thereof. 19.The method of claim 15, further comprising (i) optionally cooking andcooling soy feedstock prior to inoculating the mash and (ii) evaporatingthe separated liquid.
 20. The product of the method of claim 19.